Human application of engineered chimeric antigen receptor (car) t-cells

ABSTRACT

The present invention concerns methods and compositions for immunotherapy employing a modified T cell comprising a chimeric antigen receptor (CAR). In particular aspects, CAR-expressing T-cells are producing using electroporation in conjunction with a transposon-based integration system to produce a population of CAR-expressing cells that require minimal ex vivo expansion or that can be directly administered to patients for disease (e.g., cancer) treatment.

This application claims the benefit of U.S. Provisional PatentApplication No. 61/823,253 filed May 14, 2013, the entirety of which isincorporated herein by reference.

The invention was made with government support under Grant No.W81XWH-11-1-0002-01 awarded by the Department of Defense. The governmenthas certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTFC.P1222WO_ST25.txt”, which is 37 KB (as measured in MicrosoftWindows®) and was created on May 14, 2014, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of medicine,immunology, cell biology, and molecular biology. In certain aspects, thefield of the invention concerns immunotherapy. More particularly, itconcerns the manufacture of clinical-grade chimeric antigen receptor(CAR) T cells and therapeutic methods using such cells.

2. Description of Related Art

The potency of clinical-grade T cells can be improved by combining genetherapy with immunotherapy to engineer a biologic product with thepotential for superior (i) recognition of tumor-associated antigens(TAAs), (ii) persistence after infusion, (iii) potential for migrationto tumor sites, and (iv) ability to recycle effector functions withinthe tumor microenvironment. Such a combination of gene therapy withimmunotherapy can redirect the specificity of T cells for B-lineageantigens and patients with advanced B-cell malignancies benefit frominfusion of such tumor-specific T cells (Jena et al., 2010; Till et al.,2008; Porter et al., 2011; Brentjens et al., 2011; Cooper and Bollard,2012; Kalos et al., 2011; Kochenderfer et al., 2010; Kochenderfer etal., 2012; Brentjens et al., 2013). Most approaches to geneticmanipulation of T cells engineered for human application have usedretrovirus and lentivirus for the stable expression of chimeric antigenreceptor (CAR) (Jena et al., 2010; Ertl et al., 2011; Kohn et al.,2011). This approach, although compliant with current good manufacturingpractice (cGMP), can be expensive as it relies on the manufacture andrelease of clinical-grade recombinant virus from a limited number ofproduction facilities. New methods are needed to generategenetically-modified clinical-grade T cell products with specificity forhematologic malignancies and solid tumors.

SUMMARY OF THE INVENTION

In a first embodiment there is provided a method of providing a T-cellresponse in a human subject having a disease comprising obtaining asample of cells from the subject, (comprising T-cells or T-cellprogenitors); transfecting the cells with a nucleic acid encodingchimeric T-cell receptor (CAR), capable of integration into the genomeof the cells, and administering an effective amount of the transgeniccells to the subject to provide a T-cell response.

Thus, in some aspects, a method of the embodiments comprises: (a)obtaining a sample of cells from the subject, the sample comprisingT-cells or T-cell progenitors; (b) transfecting the cells with a DNAencoding a transposon-flanked chimeric antigen receptor (CAR) and atransposase effective to integrate the DNA encoding the CAR into thegenome of the cells, to provide a population of transgenicCAR-expressing cells; (c) optionally, culturing the population oftransgenic CAR cells ex vivo in a medium that selectively enhancesproliferation of CAR-expressing T-cells, wherein the transgenic CARcells are cultured, if at all, no more than 21 days; and (d)administering an effective amount of the transgenic CAR cells to thesubject to provide a T-cell response. Thus, in some aspects, thetransgenic CAR cells are cultured ex vivo for less than 21 days, such asfor less than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2days or less. In certain aspects, the CAR cells are cultured ex vivo nomore that 3 to 5 days. In still further aspects, steps (a)-(d) of theinstant method (i.e., obtaining cell samples to administering CAR Tcells) is completed in no more that 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, or 5 days.

In further aspects, a method of providing a T-cell response in a humansubject having a disease according to the embodiments comprises: (a)obtaining a sample of cells from the subject, the sample comprisingT-cells or T-cell progenitors and having an initial volume of betweenabout 20 and 200 mls when obtained from the subject; (b) transfectingthe cells with a DNA encoding a transposon-flanked chimeric antigenreceptor (CAR) and a transposase effective to integrate the DNA encodingthe CAR into the genome of the cells, to provide a population oftransgenic CAR-expressing T-cells; (c) optionally, culturing thepopulation of transgenic CAR cells ex vivo in a medium that selectivelyenhances proliferation of CAR-expressing T-cells; and (d) administeringan effective amount of the transgenic CAR T-cells to the subject toprovide a T-cell response. For example, the sample of cells from thesubject may be a sample of less than about 200 mls of a peripheral bloodor umbilical cord blood. In some aspects, the sample may be collected byapheresis. However, in certain preferred aspects, the sample iscollected by a method that does not involved apheresis (e.g., byvenipuncture). In still further aspects, the sample of cells has aninitial volume of less than about 175, 150, 125, 100, 75, 50 or 25 mls(e.g., the sample of cells has an initial volume of between about 50 and200 mls, 50 and 100 mls, or 100 and 200 mls when obtained from thesubject).

In a further embodiment there is provided an isolated transgenic cellcomprising an expressed CAR targeted to the envelope protein of HERV-K.In certain aspects, the cells comprise DNA encoding the CAR integratedinto the genome of the cell (e.g., CAR DNA flanked by transposon repeatsequences). For example, the CAR sequence can comprise the CDR sequences(e.g., CDRs 1-6) of monoclonal antibody 6H5 or the scFv sequence ofmonoclonal antibody 6H5. In some aspects, HERV-K-targeted CAR is atleast 85% identical to the amino acid sequence of SEQ ID NO: 4 (e.g., asequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to SEQ ID NO: 4). In certain aspects, a cell of theembodiments (e.g., a human HERV-K-targeted CAR cell) can be used totreat a subject (or provide an immune response in a subject) having aHERV-K-expressing cancer.

In still a further embodiment there is provided an isolated transgeniccell comprising an expressed CAR and an expressed membrane-bound IL-15.For example, in some aspects, the membrane-bound IL-15 comprises afusion protein between IL-15 and IL-15Rα. In yet further aspects, themembrane-bound IL-15 comprises an amino acid sequence at least about85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQID NO: 6 (referred to herein as mIL15). As further detailed herein, insome cases, the membrane-bound IL-15 is encoded by a RNA or a DNA (e.g.,an extra chromosomal or episomal vector). In certain aspects, the cellcomprises DNA encoding the membrane-bound IL-15 integrated into thegenome of the cell (e.g., coding DNA flanked by transposon repeatsequences). In certain aspects, a cell of the embodiments (e.g., humanCAR cell, expressing a membrane-bound cytokine) can be used to treat asubject (or provide an immune response in a subject) having low levelsof target antigen, such as a subject with minimal residual disease (asfurther detailed herein).

In some aspects, methods of the embodiments concern transfecting thecells with a DNA encoding a chimeric T-cell receptor (CAR) and, in somecases, a transposase. Methods of transfecting of cells are well known inthe art, but in certain aspects, highly efficient transfections methodssuch as electroporation are employed. For example, nucleic acids may beintroduced into cells using a nucleofection apparatus. Preferably, thetransfection step does not involve infecting or transducing the cellswith virus, which can cause genotoxicity and/or lead to an immuneresponse to cells containing viral sequences in a treated subject.

Further aspects of the embodiments concern transfecting cells with anexpression vector encoding a CAR. A wide range of CAR constructs andexpression vectors for the same are known in the art and are furtherdetailed herein. For example, in some aspects, the CAR expression vectoris a DNA expression vector such as a plasmid, linear expression vectoror an episome. In some aspects, the vector comprises additionalsequences, such as sequence that facilitate expression of the CAR, sucha promoter, enhancer, poly-A signal, and/or one or more introns. Inpreferred aspects, the CAR coding sequence is flanked by transposonsequences, such that the presence of a transposase allows the codingsequence to integrate into the genome of the transfected cell.

As detailed supra, in certain aspects, cells are further transfectedwith a transposase that facilitates integration of a CAR coding sequenceinto the genome of the transfected cells. In some aspects, thetransposase is provided as DNA expression vector. However, in preferredaspects, the transposase is provided as an expressible RNA or a proteinsuch that long-term expression of the transposase does not occur in thetransgenic cells. For example, in some aspects, the transposase isprovided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail).Any transposase system may be used in accordance with the embodiments.However, in some aspects, the transposase is salmonid-type Tcl-liketransposase (SB). For example, the transposase can be the so called“Sleeping beauty” transposase, see e.g., U.S. Pat. No. 6,489,458,incorporated herein by reference. In certain aspects, the transposase isan engineered enzyme with increased enzymatic activity. Some specificexamples of transposases include, without limitation, SB10, SB11 orSB100× transposase (see, e.g., Mates et al., 2009, incorporated hereinby reference). For example, a method can involve electroporation ofcells with a mRNA encoding a SB10, SB11 or SB100× transposase.

In still further aspects, a transgenic CAR cell of the embodimentsfurther comprises an expression vector for expression of amembrane-bound cytokine that stimulates proliferation and/or survival ofT-cells. In particular, CAR cells comprising such cytokines canproliferate and/or persist with little or no ex vivo culture withactivating and propagating cells (AaPCs) or artificial antigenpresenting cells (aAPCs) due the simulation provided by the cytokineexpression. Likewise, such CAR cells can proliferate in vivo even whenlarge amounts of antigen recognized by the CAR is not present (e.g., asin the case of a cancer patient in remission or a patient with minimalresidual disease). In some aspects, the CAR cells comprise a DNA or RNAexpression vector for expression of a Cγ cytokine and elements (e.g., atransmembrane domain) to provide surface expression of the cytokine. Forexample, the CAR cells can comprise membrane-bound versions of IL-7,IL-15 or IL-21. In some aspects, the cytokine is tethered to themembrane by fusion of the cytokine coding sequence with the receptor forthe cytokine. For example, a cell can comprise a vector for expressionof a IL-15-IL-15Rα fusion protein (e.g., a protein comprising thesequence of SEQ ID NO: 6). In still further aspects, a vector encoding amembrane-bound Cγ cytokine is a DNA expression vector, such as vectorintegrated into the genome of the CAR cells or an extra-chromosomalvector (e.g., and episomal vector). In still further aspects, expressionof the membrane-bound Cγ cytokine is under the control of an induciblepromoter (e.g., a drug inducible promoter) such that the expression ofthe cytokine in the CAR cells (and thereby the proliferation of the CARcells) can be controlled by inducing or suppressing promoter activity.

Aspects of the embodiments concern obtaining a sample from a patientcomprising NK cells, NKT cells, T-cells or T-cell progenitor cells. Forexample, in some cases, the sample is an umbilical cord blood sample, aperipheral blood sample (e.g., a mononuclear cell fraction) or a samplefrom the subject comprising pluripotent cells. In some aspects, a samplefrom the subject can be cultured to generate induced pluripotent stem(iPS) cells and these cells used to produce NK cells, NKT cells orT-cells. Cell samples may be cultured directly from the subject or maybe cryopreserved prior to use. In some aspects, obtaining a cell samplecomprises collecting a cell sample. In other aspects, the sample isobtained by a third party. In still further aspects, a sample from asubject can be treated to purify or enrich the T-cells or T-cellprogenitors in the sample. For example, the sample can be subjected togradient purification, cell culture selection and/or cell sorting (e.g.,via fluorescence-activated cell sorting (FACS)).

In some aspects, a method of the embodiments further comprisesobtaining, producing or using antigen presenting cells. For example, theantigen presenting cells can be dendritic cells, activating andpropagating cells (AaPCs), or inactivated (e.g., irradiated) artificialantigen presenting cells (aAPCs). Methods for producing such aAPCs areknow in the art and further detailed herein. Thus, in some aspects,transgenic CAR cells are co-cultured with inactivated aAPCs ex vivo fora limited period of time in order to expand the CAR cell population. Thestep of co-culturing CAR cells with aAPCs can be done in a medium thatcomprises, for example, interleukin-21 (IL-21) and/or interleukin-2(IL-2). In some aspects, the co-culturing is performed at a ratio of CARcells to inactivated aAPCs of about 10:1 to about 1:10; about 3:1 toabout 1:5; or about 1:1 to about 1:3. For example, the co-culture of CARcells and aAPCs can be at a ratio of about 1:1, about 1:2 or about 1:3.

In some aspects, cells for culture of CAR cells such as AaPCs or aAPCsare engineered to express specific polypeptide to enhance growth of theCAR cells. For example, the cells can comprise (i) an antigen targetedby the CAR expressed on the transgenic CAR cells; (ii) CD64; (ii) CD86;(iii) CD137L; and/or (v) membrane-bound IL-15, expressed on the surfaceof the aAPCs. In some aspects, the AaPCs or aAPCS comprise a CAR-bindingantibody or fragment thereof expressed on the surface of the AaPCs oraAPCs. Preferably, AaPCs or aAPCs for use in the instant methods aretested for, and confirmed to be free of, infectious material and/or aretested and confirmed to be inactivated and non-proliferating.

While expansion on AaPCs or aAPCs can increase the number orconcentration of CAR cells in a culture, this proceed is labor intensiveand expensive. Moreover, in some aspects, a subject in need of therapyshould be re-infused with transgenic CAR cells in as short a time aspossible. Thus, in some aspects, ex vivo culturing the transgenic CARcells (c) is for no more than 14 days, no more than 7 days or no morethan 3 days. For example, the ex vivo culture (e.g., culture in thepresence of AaPCs or aAPCs) can be performed for less than onepopulation doubling of the transgenic CAR cells. In still furtheraspects, the transgenic cells are not cultured ex vivo in the presenceof AaPCs or aAPCs.

In still further aspects, a method of the embodiments comprises a stepfor enriching the cell population for CAR-expressing T-cells aftertransfection of the cells (step (b)) or after ex vivo expansion of thecells (step (c)). For example, the enrichment step can comprise sortingof the cell (e.g., via FACS), for example, by using an antigen bound bythe CAR or a CAR-binding antibody. In still further aspects, theenrichment step comprises depletion of the non-T-cells or depletion ofcells that lack CAR expression. For example, CD56⁺ cells can be depletedfrom a culture population. In yet further aspects, a sample of CAR cellsis preserved (or maintained in culture) when the cells are administeredto the subject. For example, a sample may be cryopreserved for laterexpansion or analysis.

In certain aspects, transgenic CAR cells of the embodiments areinactivated for expression of an endogenous T-cell receptor and/orendogenous HLA. For example, T cells can be engineered to eliminateexpression of endogenous alpha/beta T-cell receptor (TCR). In specificembodiments, CAR⁺ T cells are genetically modified to eliminateexpression of TCR. In some aspects, there is a disruption of the T-cellreceptor α/β in CAR-expressing T cells using zinc finger nucleases(ZFNs). In certain aspects, the T-cell receptor αβ-chain inCAR-expressing T cells is knocked-out, for example, by using zinc fingernucleases.

As further detailed herein, CAR cells of the embodiments can be used totreat a wide range of diseases and conditions. Essentially any diseasethat involves the specific or enhanced expression of a particularantigen can be treated by targeting CAR cells to the antigen. Forexample, autoimmune diseases, infections, and cancers can be treatedwith methods and/or compositions of the invention. These includecancers, such as primary, metastatic, recurrent, sensitive-to-therapy,refractory-to-therapy cancers (e.g., chemo-refractory cancer). Thecancer may be of the blood, lung, brain, colon, prostate, breast, liver,kidney, stomach, cervix, ovary, testes, pituitary gland, esophagus,spleen, skin, bone, and so forth (e.g., B-cell lymphomas or amelanomas). In the case of cancer treatment CAR cells typically target acancer cell antigen (also known as a tumor-associated antigen (TAA)).

In still further aspects, transgenic CAR cells of the embodiments may beused to treat subject having minimal residual disease (e.g., a subjecthaving very low amounts of CAR-targeted antigen present), such as cancerpatients that are in apparent remission. Using new highly sensitivediagnostic techniques, cancer-associated antigens (or cancer cells) canbe detected in patients that do not exhibit overt cancer symptoms. Suchpatients may be treated by the instant methods to eliminate residualdisease by use of antigen-targeted CAR cells. In preferred embodiments,transgenic CAR cells for targeting of residual disease further compriseexpression of a membrane-bound proliferative cytokine, as these cellswill retain the ability to expand in vivo despite the low amount totarget antigen.

The processes of the embodiments can be utilized to manufacture (e.g.,for clinical trials) of CAR⁺ T cells for various tumor antigens (e.g.,CD19, ROR1, CD56, EGFR, CD123, c-met, GD2). CAR⁺ T cells generated usingthis technology can be used to treat patients with leukemias (AML, ALL,CML), infections and/or solid tumors. For example, methods of theembodiments can be used to treat cell proliferative diseases, fungal,viral, bacterial or parasitic infections. Pathogens that may be targetedinclude, with limitation, Plasmodium, trypanosome, Aspergillus, Candida,HSV, RSV, EBV, CMV, JC virus, BK virus, or Ebola pathogens. Furtherexamples of antigens that can be targeted by CAR cells of theembodiments include, without limitation, CD19, CD20, carcinoembryonicantigen, alphafetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen,melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2,folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met,meothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4,ERBB2, EGFRvIII, or VEGFR2. In certain aspects, method of theembodiments concern targeting of CD19 or HERV-K-expressing cells. Forexample, a HERV-K targeted CAR cell can comprise a CAR including thescFv sequence of monoclonal antibody 6H5. In still further aspects, aCAR of the embodiments can be conjugated or fused with a cytokine, suchas IL-2, IL-7, IL-15, IL-21 or a combination thereof.

In some embodiments, methods are provided for treating an individualwith a medical condition comprising the step of providing an effectiveamount of cells from the population of cells described herein, includingmore than once in some aspects, such as at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or more days apart. In specific embodiments, thecancer is lymphoma, leukemia, non-Hodgkin's lymphoma, acutelymphoblastic leukemia, chronic lymphoblastic leukemia, chroniclymphocytic leukemia, or B cell-associated autoimmune diseases.

In still yet a further embodiment there is a provided an isolated orrecombinant polypeptide comprising the a CD19-targeted CAR comprising anamino acid sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In a related embodimentan isolated or recombinant polynucleotide sequence is provided encodinga CD19-targeted CAR (e.g., encoding an amino acid sequence 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1). For example, in some aspects, the polynucleotide sequence is 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO: 3 (which encodes the CD-19-targeted CAR) or SEQ ID NO: 4 (whichencodes the CD-19-targeted CAR, expression control sequences andflanking transposon repeats). In still a further embodiment a host cellis provided comprising polypeptide encoding a CD 19-targeted CAR and/ora polynucleotide encoding a CD19-targeted CAR of the embodiments. Forexample, the host cell can be a T-cell or T-cell precursor. Preferablythe host cell is a human cell. A skilled artisan will recognize that anyof the forgoing polypeptides, polynucleotides or host cells may be usedin accordance with the methods detailed herein.

In still yet a further embodiment there is a provided an isolated orrecombinant polypeptide comprising the a HERV-K-targeted CAR comprisingan amino acid sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 4. In a related embodimentan isolated or recombinant polynucleotide sequence is provided encodinga HERV-K-targeted CAR (e.g., encoding an amino acid sequence 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4). For example, in some aspects, the polynucleotide sequence is 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO: 5 (which encodes the HERV-K-targeted CAR, expression controlsequences and flanking transposon repeats). In still a furtherembodiment a host cell is provided comprising polypeptide encoding aHERV-K-targeted CAR and/or a polynucleotide encoding a HERV-K-targetedCAR of the embodiments. For example, the host cell can be a T-cell orT-cell precursor. Preferably the host cell is a human cell. A skilledartisan will recognize that any of the forgoing polypeptides,polynucleotides or host cells may be used in accordance with the methodsdetailed herein.

In still yet a further embodiment there is a provided an isolated orrecombinant polypeptide comprising the a membrane-bound IL-15 comprisingan amino acid sequence at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 6. In a related embodimentan isolated or recombinant polynucleotide sequence is provided encodinga membrane-bound IL-15 (e.g., encoding an amino acid sequence 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6). For example, in some aspects, the polynucleotide sequence is 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO: 7 (a membrane-bound IL-15, expression control sequences and flankingtransposon repeats). In still a further embodiment a host cell isprovided comprising polypeptide encoding a membrane-bound IL-15 and/or apolynucleotide encoding a membrane-bound IL-15 of the embodiments. Forexample, the host cell can be a T-cell, T-cell precursor or aAPC.Preferably the host cell is a human cell. A skilled artisan willrecognize that any of the forgoing polypeptides, polynucleotides or hostcells may be used in accordance with the methods detailed herein.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Steps outlining the process to electroporate and propagate CAR⁺T cells from PB and UCB.

FIG. 2: Characterization of genetically modified T cells from PB. (A)Expression of EGFP at Day 0 of first stimulation cycle to assess theefficiency of gene transfer. Expression of CD19-specific CAR (CD19RCD28)as assessed by flow cytometry on CD3⁺, CD8⁺ and CD4⁺ T cells at (B)approximately 24 hours after electroporation and (C) 28 days afterco-culture on aAPC. Similar expression of CAR was observed withUCB-derived T cells. (D) Kinetics of CAR expression.

FIG. 3: Propagation of PB-derived CAR⁺ T cells. Rate of numericexpansion of CD3⁺ and CAR⁺ T cells derived from PB by repeatedco-culture on γ-irradiated aAPC in presence of recombinant human solubleIL-2 and IL-21. Upward arrows indicate the additions of γ-irradiatedaAPC that mark the beginning of each stimulation cycle. UCB-derived CAR⁺T cells exhibit similar rates of numeric expansion.

FIG. 4: Schematic of the manufacturing process using SB and aAPC systemsto genetically modify and propagate CAR⁺ T cells derived from PB andUCB. CD19-specific CAR⁺ T cells were generated by electro-transfer ofSB-derived supercoiled DNA plasmids and subsequent co-culture onK562-derived aAPC (clone #4) in the presence of recombinant humansoluble IL-2 and IL-21.

FIG. 5: Harvest and characterization of aAPC. (A, B) Sepax volumereduction. aAPC clone #4 grown in VueLife bags were harvested usingCS-490.1 kit in Sepax II. The Sepax harvest (S, n=4) was compared tomanual (M, n=1) procedure. The mean pre/post-processing cell-counts(4.9×10⁸ vs. 5×10⁸) were similar using the Sepax system. (C) Phenotypeof aAPC (clone #4). Flow cytometry analysis showing expression of CD19,CD64, CD86, CD137L and a membrane-bound version of IL-15 (peptide fusedto modified IgG4 Fc region) co-expressed with EGFP (mIL-15-EGFP) on K562aAPC and K562 parental controls.

FIG. 6: Schematic of the process of generating clinical gradeCD19-specific T cells. A MCB (PACT) and WCB (MDACC) were generated forK562-derived aAPC (clone #4). For the generation of CAR⁺ T cells, aAPCwere numerically expanded in bags, harvested using the Sepax II system,irradiated (100 Gy), and cryopreserved for later use. CD19-specific Tcells were manufactured as follows; PBMC were isolated from normal donorapheresis products using the Sepax II system and cryopreserved. The PBMCwere later thawed, electroporated with the SB DNA plasmids (CD19RCD28CAR transposon, SB11 transposase) using the Nucleofector System,co-cultured with thawed irradiated aAPC along with cytokines (IL-2 andIL-21) for a culture period of 28 days and cryopreserved.

FIG. 7: Phenotype of CAR⁺ T cell. (A) Expression of CD19RCD28 CAR on Tcells day after electroporation (culture day 1) and after 28 days ofco-culture on aAPC clone #4 along with lack of CD19⁺ aAPC. (B) CARexpression by western blot analysis using CD3-ζ specific antibody. Wholecell lysates were run on SDS-PAGE under reducing conditions. Molecularweight marker (M), Parental Jurkat cells (Lane 1), CD19RCD28⁺ Jurkatcells (Lane 2), CAR^(neg) control primary T cells (Lane 3) andCD19RCD28⁺ T cells (Lane 4). (C) Percent expression of CD3+, CD4⁺CAR⁺and CD8⁺CAR⁺ T cells with in a lymphocyte gate in cultures over time.Each symbol represents a separate experiment; the solid lines are meanof the three validation experiments. (D) Immunophenotype ofmemory/naïve, adhesion, activation, cytolytic and exhaustion markers onCAR⁺ T cells at the end (d28) of co-culture.

FIG. 8: Expansion kinetics and redirected specificity of CAR⁺ T cells.Genetically modified T cells were co-cultured with aAPC clone #4 for 28days. At the end of each stimulation cycle (7 days), cells were countedand stained for expression of CAR and CD3. Three validation runs (V1,V2, and V3) were performed and the graphs represent inferred (A) CAR⁺ Tcells, (B) CD3⁺ T cells, (C) Total viable cells over time. Arrowsindicate addition of aAPC to the culture. (D) Lysis of CD19⁺ targets(Daudiβ₂m, NALM-6, CD19⁺ EL-4) as compared to background lysis ofCD19^(neg) EL-4 using 4-hr chromium release assay by CAR⁺ T cells.Mean±SD of three validation runs is represented.

FIG. 9: Safety profile associated with the SB system. (A) Telomerelength of cells was measured using fluorescence in situ hybridizationand flow cytometry (Flow-FISH) assay. Predominant T cell population atday 28 (V1 and V2, CD8⁺ T cells; V3, CD4⁺ T cells) was compared torespective miltenyi column purified subset of T cells from day 0. (B)Genomic DNA from CAR⁺ T cells at day 28 was amplified using primers andprobes specific for CD19RCD28 CAR. Relative Quantity (RQ) analyses ofthe CD19RCD28 target copy number was determined using CD19RCD28CAR-transduced Jurkat cells, which are known to have one integration ofCD19RCD28 CAR per genome from FISH analysis, as a reference andendogenous RNaseP as a normalizer. (C) TCR VEβ analysis of day 28 andday 35 CAR⁺ T cells. Data shows mean±SD of three validation run CAR⁺ Tcells as compared to day 0 unmanipulated controls. (D) Genomic PCRshowing lack of SB11 transposase integration. Genomic DNA (20 ng) wasamplified using SB11 or GAPDH primers. CAR^(neg) control T cells (lane5) and CAR⁺ T cells (lane 7) amplified using SB11 primers; CAR^(neg)control T cells (lane 6), CAR⁺ T cells (lane 8) and Jurkat stablyexpressing SB11 (lane 4) amplified using GAPDH primers. Jurkat stablyexpressing SB11 (Jurkat/SB11-IRES2-EGFP) (lane 3) and the linearizedplasmid, pKan-CMV-SB11 (lane 2) amplified using SB11 primers was used aspositive control. (E) G-banded karyotypes of CAR⁺ T cells from the threevalidation runs reveal no structural or numeric alteration. Arepresentative spread from validation 2 is shown.

FIG. 10: Generation of CD19RCD28 CAR transposon. The CD19RCD28mz(CoOp)/pEK vector containing a codon optimized chimeric antigen receptor(CAR) and SB DNA plasmid pT-MNDU3-EGFP (Singh et al., 2008; Hollis etal., 2006) were digested with SpeI & NheI and SpeI & NruI to release CARand EGFP fragments respectively. The EGFP-deleted pT-MNDU3 vector wasthen ligated with CAR fragment to generate CD19RCD28mz (CoOp)/pT-MNDU3vector. Further the Kanamycin resistance gene and the ColE1 origin ofreplication obtained by AseI & PacI digestion of pEK vector was ligatedinto SalI & ZraI digested CD19RCD28mz (CoOp)pT-MNDU3 vector to createPre-CD19RCD28mz (CoOp)/pSBSO. In the last step, MNDU3 promoter fromPre-CD19RCD28mz(CoOp)/pSBSO was released using digestion with NheI &NsiI and replaced with hEF-1a promoter fragment obtained from pVitro4vector using XhoI & NheI, to generate the final vectorCD19RCD29mz(CoOp)/pSBSO.

FIG. 11: Generation of SB11 transposase. SB transposase vector pCMV-SB11was digested with PvuII to release the fragment containing CMVpromoter/enhancer and SB transposase encoding gene, which was ligated tofragment containing Kanamycin resistance gene and ColE1 origin ofreplication from pEK vector to generate pKan-CMV-SB11 vector.

FIG. 12: Schematic of CD19 expression plasmid, ΔCD19CoOp-F2A-Neo/pSBSO.The DNA fragment encoding CD19RCD28 CAR from the plasmidCoOpCD19RCD28/pSBSO was swapped with DNA fragment encoding neomycinresistance gene (NeoR) [PCR cloned from pSelect-Neo (InvivoGen)] fusedto codon-optimized (GENEART) truncated CD19 (ΔCD19, [Serrano et al.,2006; Mahmoud et al., 1999]) via a F2A linker (amino acid,VKQTLNFDLLKLAGDVESNPGP; [Szymczak et al., 2004; Yang et al., 2008; Kimet al., 2011]) to generate ΔCD19CoOp-F2A-Neo/pSBSO. EFloi promoter,Elongation factor-1α promoter; NeoR, Neomycin resistance gene; bGHpAn,polyadenylation signal from bovine growth hormone; ColE1, ori; KanR,Kanamycin resistance gene; IR, SB-inverted/direct repeats.

FIG. 13: Rate of numeric expansion of CD19-specific CAR⁺ T cells.Genetically modified T cells were co-cultured with aAPC in a 7-daystimulation cycle, and weekly fold-expansion rate from each validationrun at the end of each stimulation cycle for total, CD3⁺ and CAR⁺ Tcells was calculated. Mean fold-expansion is shown (n=3).

FIG. 14: Redirected specificity of CD19-specific CAR⁺ T cells.CD19-specific lysis of CD19⁺ tumor targets (Daudiβ₂m, NALM-6, CD19⁺EL-4) by CAR+ T cells generated in three validation runs (V1, V2, V3) ina standard 4-hr chromium assay. Background autologous lysis against theCAR^(neg) control was 1.5%.

FIG. 15: Safety regarding chromosomal aberration. G-banded karyotypes ofCAR⁺ T cells generated from validation runs (V1 and V3) reveal nostructural or numeric alteration.

FIG. 16: (A) A representative picture of tumor cells (200×) with varyingintensity (Scored 0-3) of HERV-K expression (top panel) when compared toisotype IgG2a control staining (bottom panel). (B) A picture of tumorcells (400×) showing HERV-K staining that is punctate and bordering thecell membrane (solid arrow) or more diffuse cytoplasmic staining (dottedarrow). (C) A dot plot representing H-index of each patient showed asignificant difference (p<0.0267) between the benign and tumor tissues.(D) No significant difference was seen between malignant and metastatictumor. And significant difference was seen between the benign tumor andmalignant or metastatic tumor.

FIG. 17: (A) HERV-K-specific CAR encoding SB plasmid. (B) Flow plotrepresenting CAR (Fc) expression on Day 1 and Day 35 of CD3⁺HERV-K-specific CAR⁺ T cells. Quadrant percentages of flow plots are inupper right corner. CAR, chimeric antigen receptor. (C) No significantdifference in total cell growth between the HERV-K-specific CAR⁺ T cellsand non-specific CD-19 CAR⁺ T cells. (D) By Day 21 all HERV-K-specificCAR cells are CD3⁺ T cells. (E) The CAR integration analysis shows thatthe HERV-K-specific CAR⁺ T cells have less than 2 integrations per cell.Data represents mean of two independent experiments with 3 differentdonors performed in triplicate. (F) The phenotype of HERV-K-specificCAR⁺ T cells are CD3⁺CD56⁺CD45RO^(hi) CD45RA^(lo)CD27⁺CD62L⁺ T cellsthat produce high levels of granzyme B. All data represent average offour donors.

FIG. 18: (A) A histogram representation on HERV-K antigen expression (inred) on tumor cell surface compared to isotype control (in blue). (B) Astandard 4-h CRA of melanoma tumor targets with varying dilution ofHERV-K-specific CAR⁺ T cells (in solid line) compared to No DNA controlT cells (in dotted lines). Data are mean±SD from four healthy donors(average of triplicate measurements for each donor) that were pooledfrom two independent experiments. Two-way ANOVA with Bonferronipost-test was performed on (B) and (C) between the HERV-K-specific CAR⁺T cells and No DNA control cells. CAR, chimeric antigen receptor; CRA,chromium release assay; E:T, effector to target ratio. (D) IFN-γproduction by CAR⁺ T cells upon incubation with targets. PMA-ionomycinis used as a positive control.

FIG. 19: Specificity of HERV-K-specific CAR⁺ T cells. (A) Histogram ofEL4 cells artificially expressing HERV-K antigen (in black) was plottedalong with HERV-K^(neg) EL4 parental (blue) and isotype control staining(orange). (B) A four hour CRA showed a significant increase (p<0.001) inkilling EL4 cells expressing the antigen compared to the parental by theHERV-K-specific CAR⁺ T cells at varying E:T ratios. (C) Immunoblot assaywas performed to show HERV-K env-specific shRNA-mediated knockdown inA888 cells when compared to A888 parent or A888 treated with scrambledshRNA. Lower panel shows actin protein expression as control. (D) CRA ofHERV-K-specific CAR⁺ T cells with the A888 HERV-K KD cells, A888parental (A888P) and A888 scrambled control (A888 sera) showedsignificant antigen-specific killing by the T cells. All data representthe mean of two independent experiments by three donors preformed intriplicate. Two-way ANOVA with Bonferroni post-test was used for (B) tocompare EL4 parental to HERV-K⁺ EL4 and one-way ANOVA with Newman-Keulsmultiple comparison test for (C) to compare A888KD to A888 P and A888sera.

FIG. 20: To determine the activity of HERV-K-specific CAR⁺ T cells over15 h period target and effector cells were plated in 1:5 ratio withSytox® (Invitrogen, dead cell stain) in the media. Fifty images of eachtarget with effector cells were recorded every 7 min for this period.(A) Picture representing HERV-K⁺ melanoma cells (A888 and A375) andHERV-Kneg control (HEK293 parent) cells with CAR⁺ T cells are varioustime points. Cells that turned green were recorded as dead cells and theintensity of the fluorescence was measured. (B, C, D) Represents themean fluorescent intensity of the target cell. Upper lines represent thedead cells while the lower lines represent the basal intensity of livecells. (E) A plot representing a significant difference (*p<0.05) in themean fluorescent intensity at 15 h time point compared to HEK293parental cells. Data represents average of 2 independent experimentswith 50 images each. A one-way ANOVA with Tukey's post-test wasperformed.

FIG. 21: In vivo antitumor activity of HERV-K-specific CAR⁺ T cells. (A)Schematic of experiment. (B) Representative images of mice from day 3 today 25. (C) BLI derived from mKate⁺rRLuc⁺HERV-K⁺ A375-SM tumor and (D)postmortem analysis of liver tissues with the red dots representingmKate⁺tumor metastatic foci. Data are mean±SD (n=5-6 mice per group).Statistics performed with (in D) two-way ANOVA with Bonferroni'spost-tests and between treated and untreated mice. **P<0.01 and***P<0.001. ANOVA, analysis of variance; BLI, bioluminescent imaging;CAR, chimeric antigen receptor; IL, interleukin.

FIG. 22: (A) Representative pictures (200×) of HERV-K antigen expressionon tissues sections from 29 normal organs are shown. The H-index wascalculated as zero since no staining was observed in any of thesetissues. (B) H-index of malignant tissue from various organs anddifferent patient are shown in a dot plot.

FIG. 23: (A) Growth of CD4⁺ versus CD8⁺ HERV-K-specific CAR⁺ T cells isshown. (B) nCounter analysis representing expression of various genes inHERV-K-specific CAR⁺ T cells versus No DNA control cells. Red indicateshigh expression while green indicates low mRNA levels. (C) Ingenuitypathway analysis of genes highly expressed in HERV-K-specific CAR⁺ Tcells.

FIG. 24: A 4 h standard CRA was performed with melanoma andCD19-specific tumor targets and CD19 CAR⁺ T cells. All data representtwo independent experiments performed with an average of 6 donors andanalyzed using 2-way ANOVA with Bonferroni post-test.

FIG. 25: Bi-directional SB plasmid encoding HERV-K antigen under hEF-1αpromoter and neomycin resistance gene under CMV promoter.

FIG. 26: Figure representing HERV-K-specific CAR (in green) engagementwith HERV-K antigen (in red) on tumor cell surface.

FIG. 27: (A) SB Plasmid encoding myc-ffLuc with Neomycin resistancegene. (B) Total cell growth of HERV-K-specific CAR⁺ T cells andHERV-K-specific CAR-ffLuc⁺ T cells. (C) Four-hour CRA of A375SM and EL4parental cells with HERV-K-specific CAR-ffLuc⁺ T cells. (D) Mouse imagerepresenting ffLuc activity. (E) Lentiviral plasmid encoding RLuc andmKate for tumor cell imaging.

FIG. 28A-B: Schematics of mIL15. A) The mIL15 construct flanked byinverted repeats which are components of the Sleeping Beauty expressionplasmid. The mIL15 mutein is a fusion of IL-15 with the full-lengthIL-15Rα by a flexible serine-glycine linker. B) A schematic representingthe expressed protein structure of mIL15.

FIGS. 29A-B: CAR and mIL15 expression in genetically modified T cellsafter ex vivo expansion on aAPC after five stimulation cycles. A)Expression of a representative sample of five donors. B) Expression ofthe denoted marker (CAR and/or mIL15) in genetically modified T cells.

FIG. 30: Validation of the functionality of mIL15 via phosflow ofpSTAT5. A five hour incubation of cells in serum and cytokine-freeconditions, unless otherwise noted, to obtain basal and IL-15-mediatedphosphorylation. Representative plot (n=6).

FIG. 31: Inferred counts of CAR⁺ T cells with or without co-expressionof mIL15 after four stimulation cycles on aAPC. CAR⁺ T cells werecultured with soluble IL-2 and IL-21 (the standard culture condition) orIL-15 and IL-21 (the soluble cytokine control), and mIL15⁺CAR⁺ T cellswith IL-21. Data are mean±SD, n=4.

FIGS. 32A-B: Phenotype and specific lysis capacity of ex vivo expandedmIL15⁺CAR⁺ T cells. A) Percent surface expression of certain T cell,activation, and differentiation-associated markers after 4 stimulationson aAPC. Horizontal line indicates the mean value. *P=0.047, paired ttest, n>4. B) CAR⁺ T cell (left panel) and mIL15⁺CAR⁺ T cell (rightpanel) specific lysis after 5 stimulations on aAPC of CD19⁺ orCD19^(neg) targets from a four-hour chromium release assay. Data arerepresented as mean±SD, n=3.

FIG. 33: In vitro long-term persistence of mIL15⁺CAR⁺ T cells thatremain functionally competent and resistant to AICD. A) Ex vivo expandedmIL15^(+/−)CAR⁺ T cells after four aAPC stimulations underwentwithdrawal from antigen re-stimulation to assess long-term in vitropersistence and observe expansion kinetics over 60+ days. The mIL15⁺CAR⁺T cells did not receive any exogenous cytokine support, whereas CAR⁺ Tcells received no cytokines, IL-2, or IL-15. Data are log of mean±SD,****P<0.0001, RM ANOVA, n=3. B) The surviving T cells at greater than 75days post-antigen exposure were tested for antigen responsiveness byincubation with: no target, CD19^(+/−) EL4, CD19⁺ Nalm-6, or LAC for 6hours. Analysis was by flow cytometry of IFNγ intracellular staining.Representative flow plots shown, n=3. C) The surviving T cells after 75days post-withdrawal were tested stimulated 1:1 with aAPC as previouslydescribed and media was supplemented with the cytokine (if any) usedduring the withdrawal culture maintenance plus the addition of IL-21.After eight days, T cells were stained with Annexin V to determine theproportion of live versus apoptotic/necrotic cells in the stimulatedculture. Representative flow plot shown, n=3.

FIGS. 34A-C: Long-term persisting mIL15⁺CAR⁺ T cells take on dichotomousCD45RA⁺CCR7^(+/−) phenotypes. A) Representative flow plots of CD45RA andCCR7 populations of mIL15⁺CAR⁺ T cells from stimulation 4 and thosepersisting 75 days after last antigen stimulation, n=7. B) Frequenciesof populations subsets from (A). ***P<0.001 and ****P<0.0001, RM ANOVA,n=7. C) A representative histogram showing Annexin V levels inCCR7^(neg) and CCR7⁺ mIL15⁺CAR⁺ T cells from stimulation 4 and thosepersisting 75 days after last antigen stimulation, n=3. Histograms aregated on the lymphocyte population to avoid non-specific CCR7 staining

FIG. 35A-D: Graphs show additional characterization of mIL15⁺CAR⁺ Tcells by flow cytometry analysis. FIG. 35A, Homeostatic proliferationlevel of WD-mIL15⁺CAR⁺ T cells from three normal donors (PKH dilution at10 days after staining) that have been in culture without antigenre-stimulation for 1-2 years (top panel) and proliferative capacity ofthese cells upon antigen re-stimulation with aAPC (bottom panel). FIG.35B, Phenotype of long-term withdrawal mIL15⁺CAR⁺ T cells (CD3 and mIL15surface expression) that were submitted for karyotyping. Withdrawal Tcells were first re-stimulated with aAPC prior to phenotyping andsubmission for karyotyping. FIG. 35C, Normal karyotype (G-banding)result of mIL15⁺CAR⁺ T cells persisting long-term (1.5-2.46 years) invitro in the absence of antigen re-stimulation and exogenous cytokines.A representative metaphase spread is shown from four normal donors. FIG.35D, Memory kinetics after stimulation of cells using K562 aAPCs. FIG.3E, Memory kinetics with 1 versus 2 stimulations of CAR and mIL15⁺CAR⁺ Tcells.

FIG. 36: Molecular profiling indicates long-term persisting mIL15⁺CAR⁺ Tcells exhibit characteristics associated with less differentiated T cellsubsets. All genes significantly differentially expressed betweenmIL15⁺CAR⁺ T cells from stimulation four and those persisting throughwithdrawal conditions are functionally classified under broad categoriesbased on gene ontology information. Genes within categories arepartitioned based on up- or down-regulation in the persisting withdrawalmIL15⁺CAR⁺ T cells.

FIG. 37: Validation of transcription factors associated with T celldifferentiation states indicates long-term persisting mIL15⁺CAR⁺ T cellsexhibit a low differentiation state. Top panel: Selected differentiallyexpressed genes (Tcf-7, Blimp-1, and T-bet) were validated byintracellular staining and analyzed by flow cytometry. Representativeflow plots shown, n=5. Bottom panel: Normalized mRNA copy number fromthe nCounter Analysis System output.

FIG. 38: Validation of surface markers associated with T celldifferentiation states indicates long-term persisting mIL15⁺CAR⁺ T cellsexhibit less differentiation. Top panel: Selected differentiallyexpressed genes, IL-7Ra and CCR7, were validated by staining andanalyzed by flow cytometry. *P=0.0156 and ***P<0.001, 1-tailed Wilcoxonmatched-pairs signed rank test and paired 1-tailed T test, respectively.Representative flow plots shown, n=5-7. Bottom panel: Normalized mRNAcopy number from the nCounter Analysis System output, n=3.

FIG. 39: Acquisition of IL-2 production capability by mIL15⁺CAR⁺ Tcells. A) Representative histograms of IL-2 intracellular staining ofstimulation 4 and persisting antigen withdrawal mIL15⁺CAR⁺ T cells(WD-mIL15-CAR) that were either unstimulated or activated withlymphocyte activation cocktail (LAC) for 6 hours, followed byintracellular IL-2 staining and analysis by flow cytometry. B) Frequencyof LAC-stimulated T cells producing IL-2 from (A). ****P<0.0001, n=6,paired t test.

FIGS. 40A-D: In vivo persistence and anti-tumor activity of mIL15⁺CAR⁺ Tcells in an environment with abundant tumor antigen. A) Schematic ofexperiment. B) BLI derived from mIL15^(+/−)CAR⁺ffluc⁺ T cells adoptivelytransferred into mice after Nalm-6 tumor introduction (n=5). C) Analysisof spleen (top panel) and bone marrow (bottom panel) at day 14 for thepresence of human T cells by staining with human CD3 and detection byflow cytometry. Representative flow plot (n=5). D) Analysis ofperipheral blood by flow cytometry for the presence of CD3⁺ and CD19⁺cells. Frequencies obtained after gating out murine CD45⁺ cells. Datadepicts individual mice and mean±SD. ***P<0.001, 1-way ANOVA, n=3-5.

FIGS. 41A-E: In vivo model assessing mIL15⁺CAR⁺ T cell persistence andanti-tumor efficacy in a low antigen environment. A) Schematic ofexperiment. B) T cell (ffLuc⁺) BLI of mice receiving adoptive transferof either CAR⁺ T cells or mIL15⁺CAR⁺ T cells followed by CD19⁺ Nalm-6tumor injection after 6 days of T cell engraftment. C) Longitudinal BLImonitoring Nalm-6 burden. Images represent photon flux from Nalm-6cell-derived rLuc activity. D) Tumor flux (rLuc) over time of micetreated with either CAR⁺ T cells, mIL15⁺CAR⁺ T cells, or no T cells.Data are mean±SD. ****P<0.0001, one-way ANOVA, n=4-5. E) Analysis ofharvested tissues and blood where human T cells (human CD3+) and Nalm-6tumor cells (human CD19⁺) were detected by flow cytometry. E) Using thelow tumor model, long-term survival of mice was evaluated out to day 98.Experimental conditions were carried out similarly as previouslydescribed for the low tumor model where mice were engrafted withmIL15-CAR T cells (n=7), CAR T cells (n=8), or no T cells (n=8) followedby NALM-6 tumor challenge. Fractions in parentheses represent theproportion of mice surviving to day 98. *P=0.045 (mIL15-CAR versus CAR Tcell treatment), log-rank (Mantel-Cox).

FIGS. 42A-E: Persistence and retained function of mIL15+CAR+ T cellsindependent of antigen. A) Schematic of experiment. B) BLI of ffLuc+ Tcells in mice treated with CAR+ and mIL15+CAR+ T cells in the absence oftumor antigen. C) Analysis of bone marrow, spleen, and peripheral bloodfor human CD3+ T cells and CD19+ tumor cells, as detected by flowcytometry. Representative flow plots are shown (left panels) and plottedfrequencies of human CD3+ T cells (right panel). Data are represented asmean±SD, n=5. **P=0.0027 (bone marrow), **P=0.0081 (spleen), ns=notsignificant, unpaired t test. D) Longitudinal plotting of T cell flux(ffluc). Background luminescence (gray shaded) was defined by fluxobtained from mice not receiving ffluc+ T cells. Data are represented asmean±SD, n=5. *P=0.0128, **P+0.00231, unpaired t test. E) Cells isolatedfrom spleen, liver, or bone marrow were ex vivo expanded on aAPC togenerate sufficient cell numbers for assessing intracellular IFNγproduction in response to CD19− and CD19+ targets (as previouslydescribed), n=7 from three tissue sources and four mice. Histograms weregated on CD3.

FIG. 43: A schematic showing an example protocol for CAR T cellproduction using SB transposase provided as a mRNA. Effective quantitiesof active CAR T cells could be produced in two weeks or less (e.g., 16days).

FIG. 44A-D: FIG. 44A, graph (left panel) and flow cytometry histograms(right panel) show the percentage of T-cells stably expressing CAR isincreasing significantly from day 9 (8.3%) to day 16 (66.2%). FIG. 44B,graph shows that T-cells grow quickly and amplify by 20-folds to day 16after electroporation. FIG. 44C, graphs show results from chromiumrelease assays using day 16 T-cells. The CAR T-cell produced providedCD-19-specific cytotoxicity against target cells (left panel).Essentially no cytotoxic activity was seen for unmodified T-cells (rightpanel). FIG. 44D, Flow cytometry histograms showing the number ofcentral memory T-cells (Tcm) at day 9 (left panel) and day 17 (rightpanel) post electroporation. Cells for these studies were from a donordesignated as #0 and SB11 mRNA was used in the electroporation.

FIG. 45A-B: FIG. 45A, graphs show results from chromium release assaysusing day 9 (upper panels) and day 15 (lower panels) T-cells. The CART-cell produced provided CD-19-specific cytotoxicity against targetcells (left panels). Essentially no cytotoxic activity was seen forunmodified T-cells (right panels). The modified T-cells killCD19-positive target cells on day 9 and day 15 with similar efficiencydespite the different number of CAR-positive cells. FIG. 45B, Graphsshow CAR copy number (left panel) and CAR expression (right panel) inelectroporated cells. Results show that CAR DNA copy number decreasesfrom 1.5 to 0.9 from day 15 to day 22 and stays stable after that timepoint. Cells for these studies were from a donor designated as #1 andSB100× mRNA was used in the electroporation.

FIG. 46: Flow cytometry data showing cell viability followingelectroporation. After electroporation with DNA/mRNA the total number ofcells decreases first (day 1 and 2) and then cells start to grow.According to the cellometer counts the cell number decrease by 59%-76%on day 2 after electroporation (viability 24-41%).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Clinical trials have demonstrated anti-tumor effects in patients thathave received T cells genetically modified to have desired specificity.Herein, a new approach to manufacturing antigen-specific CAR⁺ T cells incompliance with cGMP is detailed. The system employs a highly efficienttransfection system in conjunction with a transposon and transposasesystem CAR gene integration. These non-viral approaches have significantadvantages as an alternative to viral-mediated transduction sinceclinical grade CAR⁺ T cells preferably should not include added viralsequences. High quality cGMP CAR T-cells were achieved byelectro-transfer of DNA plasmids derived from a transposon system, suchas from Sleeping Beauty or piggyBac, and propagation of the geneticallymodified T cells on aAPC. The approach resulted in outgrowth ofclinically appealing numbers of CAR⁺ T cells that demonstratedspecificity for their target antigen (e.g., CD19). Importantly, thecells met release criteria established by the FDA for use in clinicaltrials. These methods avoid genotoxicity due to virus-mediatedtransduction and immunogenicity due to use of virus.

A chimeric antigen receptor (CAR) recognizes cell-surfacetumor-associated antigen independent of human leukocyte antigen (HLA)and employs one or more signaling molecules to activate geneticallymodified T cells for killing, proliferation, and cytokine production(Jena et al., 2010). Adoptive transfer of T cells expressing CAR hasshown promise in multiple clinical trials. It is now possible to use amodular approach to manufacture clinical grade genetically modified Tcells. In certain embodiments, the platform technologies disclosedherein comprise (i) non-viral gene transfer using an electroporationdevice (e.g., a nucleofector), (ii) transposition (e.g., an SBtransposon, see, U.S. Pat. No. 6,489,458, incorporated herein byreference), (iii) CARs that signal through endodomains (e.g.,CD28/CD3-ζ, CD137/CD3-ζ, or other combinations), (iv) CARs with variablelengths of extracellular domains connecting the antigen-recognitiondomain to the cell surface, and, in some cases, (v) artificial antigenpresenting cells (aAPC) derived from K562 to be able to robustly andnumerically expand CAR⁺ T cells (Singh et al., 2008; Singh et al., 2011;Huang et al., 2012).

In some embodiments, the presently disclosed process can be used togenetically modify T cells derived from peripheral blood and/orumbilical cord blood to express CAR(s) that can be numerically expandedin vitro using aAPC (Singh et al., 2008; Singh et al., 2011). Theprocess has implications for cell and gene therapy, due to the relativeease of DNA plasmid production, electroporation, use of thawedγ-irradiated master-bank aAPC, and can be readily transferred tofacilities operating in compliance with current good manufacturingpractice (cGMP) for Phase I/II trials. The disclosed method ofmanufacturing T cells is unique at least in that it does not use (i)viral-transduction or (ii) naked DNA electroporation followed by rapidexpansion on a PBMC/LCL feeder layer. As an example, methods aredisclosed for targeting CD19 through the enforced expression of a CARthat recognizes CD19 independent of HLA. These T cells meet releasecriteria defined by sterility, phenotype, viability, and cell number.In-process testing revealed that the electroporated/propagated T cellsexpress CAR in a memory/naïve population, have a normal karyotype,preserved TCR V13 repertoire, and are able to recognize and lyse CD19⁺tumor targets in a CAR-dependent manner.

The electro-transfer of non-viral plasmids is an appealing alternativeto transduction since DNA species can be produced to clinical grade atapproximately 1/10^(th) the cost of recombinant GMP-grade virus. Toimprove the efficiency of integration, the inventors adapted SleepingBeauty (SB) transposon and transposase for human application (Aronovichet al., 2011; Hackett et al., 2010; Izsvak et al., 2010; Kebriaei etal., 2012; Williams, 2008). Additionally, they have used the piggyBactransposon/transposase system to enforce expression of CAR (Manuri etal., 2010). The inventor's SB system uses two DNA plasmids that comprisea transposon coding for a gene of interest (e.g., 2^(nd) generationCD19-specific CAR transgene, such as designated CD19RCD28) and atransposase (e.g., SB11), which inserts the transgene into TAdinucleotide repeats in the target cell genome (Geurts et al., 2006;Ivics et al., 1997; Izsvak and Ivics, 1997). To improve therapeuticpotential, the inventor's 2^(nd) generation CAR (Kowolik et al., 2006)signals through CD28 and CD3-ζ with the expectation that this willsustain T-cell proliferation and recycle effector functions in vivo. Inaddition, CARs with varying extracellular lengths and differentendodomain signaling motifs can be expressed using the SB system.

To retrieve T-cell integrants stably expressing the CAR, K562 aAPC(clone #4) were developed, expressing the desired antigen (e.g., CD19)along with costimulatory molecules, such as CD86, CD137L, amembrane-bound version of interleukin (IL)-15 (peptide fused to modifiedIgG4 Fc region or cytokine peptide fused to IL-15 receptor alpha), andCD64 (Fc-γ receptor 1), to select for T cells in vitro that are capableof sustained CAR-mediated propagation. This powerful technology hasallowed the manufacture of clinically relevant numbers (up to 10¹⁰) ofCAR⁺ T cells suitable for human application. As needed, additionalstimulation cycles can be undertaken to generate larger numbers ofgenetically modified T cells. Furthermore, if fewer CAR⁺ T cells areneeded, the approach of electroporation and propagation can be scaledback employing fewer cuvettes and carrying forward just a sub-set of thenumerically expanded T cells for 0, 1, or more rounds of proliferationon aAPC (added at the beginning of each stimulation cycle). Typically,at least 90% of the propagated T cells express CAR and are cryopreservedfor infusion. Furthermore, this approach can be harnessed to generate Tcells to diverse tumor types by pairing the specificity of theintroduced CAR with expression of the tumor-associated antigen (TAA)recognized by the CAR on the aAPC. The ex vivo expansion platform hasalso been adapted to manufacture NK cells, NK T cells, and γδ T cells.

The outgrowth of CD4⁺ and CD8⁺ T cells expressing the 2^(nd) generationCAR include cells with a stem-cell/memory/naive phenotype and exhibitthree hallmarks of re-directed specificity. First, the geneticallymodified T cells specifically lyse CD19⁺ targets. Second, they producecytokine (e.g., IFN-γ) in response to CD19⁺ stimulator cells. Third,they proliferate in response to CD19⁺ stimulation, all in aCAR-dependent manner (Singh et al., 2011; Singh et al. 2008). The aAPCand tissue culture environment (e.g., the addition of IL-21) have beenmodified to generate patient- and donor-derived CD19-specific T cellsfor infusion after hematopoietic stem-cell transplantation (Singh etal., 2011; Singh et al. 2008). The inventors can produce CAR⁺ T cellsfrom peripheral blood simply obtained by venipuncture, which avoids thecost, discomfort, and inconvenience of obtaining mononuclear cells byapheresis. The ability to derive large numbers of CAR⁺ T cells fromsmall numbers of mononuclear cells is particularly appealing forinfusing T cells after allogeneic umbilical cord blood transplantation.The small size and anonymity of the neonatal donor precludesre-accessing this individual at a later time point and only limitednumbers of harvested mononuclear cells are available as startingmaterial for T cell manufacture to avoid interfering with hematopoiesis.Further advances to the manufacturing process include a high throughputelectroporation device coupled with a fully closed WAVE bioreactor tominimize handling.

The present approach to manufacturing includes the implementation ofprocessing and culturing systems to reduce work load and safeguardagainst a breach in sterility. To this end, the inventors co-cultured Tcells with γ-irradiated aAPC in bioreactors and/or bags rather thanflasks. This transition typically occurs after day 14 followingelectroporation. In addition, the aAPC as source material arenumerically expanded in bioreactors and/or bags, and the inventorsadapted the Sepax device to process the aAPC for cryopreservation. TheSepax harvest procedure has additional advantages beyond automation whenusing large volumes of culture media (>900 mL) as it reduces additionalcentrifuging steps required by the manual process.

I. DEFINITIONS

The term “chimeric antigen receptors (CARs),” as used herein, may referto artificial T-cell receptors, chimeric T-cell receptors, or chimericimmunoreceptors, for example, and encompass engineered receptors thatgraft an artificial specificity onto a particular immune effector cell.CARs may be employed to impart the specificity of a monoclonal antibodyonto a T cell, thereby allowing a large number of specific T cells to begenerated, for example, for use in adoptive cell therapy. In specificembodiments, CARs direct specificity of the cell to a tumor associatedantigen, for example. In some embodiments, CARs comprise anintracellular activation domain, a transmembrane domain, and anextracellular domain comprising a tumor associated antigen bindingregion. In particular aspects, CARs comprise fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies, fused toCD3-zeta a transmembrane domain and endodomain. The specificity of otherCAR designs may be derived from ligands of receptors (e.g., peptides) orfrom pattern-recognition receptors, such as Dectins. In particularembodiments, one can target malignant B cells by redirecting thespecificity of T cells by using a CAR specific for the B-lineagemolecule, CD19. In certain cases, the spacing of the antigen-recognitiondomain can be modified to reduce activation-induced cell death. Incertain cases, CARs comprise domains for additional co-stimulatorysignaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and/or OX40.In some cases, molecules can be co-expressed with the CAR, includingco-stimulatory molecules, reporter genes for imaging (e.g., for positronemission tomography), gene products that conditionally ablate the Tcells upon addition of a pro-drug, homing receptors, chemokines,chemokine receptors, cytokines, and cytokine receptors.

The term “T-cell receptor (TCR)” as used herein refers to a proteinreceptor on T cells that is composed of a heterodimer of an alpha (α)and beta (β) chain, although in some cells the TCR consists of gamma anddelta (γ/δ) chains. In embodiments of the invention, the TCR may bemodified on any cell comprising a TCR, including a helper T cell, acytotoxic T cell, a memory T cell, regulatory T cell, natural killer Tcell, and gamma delta T cell, for example.

The terms “tumor-associated antigen” and “cancer cell antigen” are usedinterchangeably herein. In each case, the terms refer to proteins,glycoproteins or carbohydrates that are specifically or preferentiallyexpressed by cancer cells.

II. CHIMERIC ANTIGEN RECEPTORS

As used herein, the term “antigen” is a molecule capable of being boundby an antibody or T-cell receptor. An antigen is additionally capable ofinducing a humoral immune response and/or cellular immune responseleading to the production of B and/or T lymphocytes.

Embodiments of the present invention involve nucleic acids, includingnucleic acids encoding an antigen-specific chimeric antigen receptor(CAR) polypeptide, including a CAR that has been humanized to reduceimmunogenicity (hCAR), comprising an intracellular signaling domain, atransmembrane domain, and an extracellular domain comprising one or moresignaling motifs. In certain embodiments, the CAR may recognize anepitope comprised of the shared space between one or more antigens.Pattern recognition receptors, such as Dectin-1, may be used to derivespecificity to a carbohydrate antigen. In certain embodiments, thebinding region can comprise complementary determining regions of amonoclonal antibody, variable regions of a monoclonal antibody, and/orantigen binding fragments thereof. In another embodiment, thatspecificity is derived from a peptide (e.g., cytokine) that binds to areceptor. A complementarity determining region (CDR) is a short aminoacid sequence found in the variable domains of antigen receptor (e.g.,immunoglobulin and T-cell receptor) proteins that complements an antigenand therefore provides the receptor with its specificity for thatparticular antigen. Each polypeptide chain of an antigen receptorcontains three CDRs (CDR1, CDR2, and CDR3). Since the antigen receptorsare typically composed of two polypeptide chains, there are six CDRs foreach antigen receptor that can come into contact with the antigen—eachheavy and light chain contains three CDRs. Because most sequencevariation associated with immunoglobulins and T-cell receptors are foundin the CDRs, these regions are sometimes referred to as hypervariabledomains. Among these, CDR3 shows the greatest variability as it isencoded by a recombination of the VJ (VDJ in the case of heavy chain andTCR αβ chain) regions.

It is contemplated that the human CAR nucleic acids are human genes toenhance cellular immunotherapy for human patients. In a specificembodiment, the invention includes a full length CAR cDNA or codingregion. The antigen binding regions or domain can comprise a fragment ofthe V_(H) and V_(L) chains of a single-chain variable fragment (scFv)derived from a particular human monoclonal antibody, such as thosedescribed in U.S. Pat. No. 7,109,304, incorporated herein by reference.The fragment can also be any number of different antigen binding domainsof a human antigen-specific antibody. In a more specific embodiment, thefragment is an antigen-specific scFv encoded by a sequence that isoptimized for human codon usage for expression in human cells.

The arrangement could be multimeric, such as a diabody or multimers. Themultimers are most likely formed by cross pairing of the variableportion of the light and heavy chains into what has been referred to byWinters as a diabody. The hinge portion of the construct can havemultiple alternatives from being totally deleted, to having the firstcysteine maintained, to a proline rather than a serine substitution, tobeing truncated up to the first cysteine. The Fc portion can be deleted.Any protein that is stable and/or dimerizes can serve this purpose. Onecould use just one of the Fc domains, e.g., either the CH2 or CH3 domainfrom human immunoglobulin. One could also use the hinge, CH2 and CH3region of a human immunoglobulin that has been modified to improvedimerization. One could also use just the hinge portion of animmunoglobulin. One could also use portions of CD8alpha.

The intracellular signaling domain of the chimeric receptor of theinvention is responsible for activation of at least one of the normaleffector functions of the immune cell in which the chimeric receptor hasbeen placed. The term “effector function” refers to a specializedfunction of a differentiated cell. Effector function of a T cell, forexample, may be cytolytic activity or helper activity including thesecretion of cytokines. Effector function in a naive, memory, ormemory-type T cell includes antigen-dependent proliferation. Thus theterm “intracellular signaling domain” refers to the portion of a proteinthat transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain will be employed, in many cases it will not benecessary to use the entire intracellular polypeptide. To the extentthat a truncated portion of the intracellular signaling domain may finduse, such truncated portion may be used in place of the intact chain aslong as it still transduces the effector function signal. The termintracellular signaling domain is thus meant to include any truncatedportion of the intracellular signaling domain sufficient to transducethe effector function signal. Examples include the zeta chain of theT-cell receptor or any of its homologs (e.g., eta, delta, gamma, orepsilon), MB 1 chain, B29, Fc RIII, Fc RI, and combinations of signalingmolecules, such as CD3ζ and CD28, CD27, 4-1BB, DAP-10, OX40, andcombinations thereof, as well as other similar molecules and fragments.Intracellular signaling portions of other members of the families ofactivating proteins can be used, such as FcγRIII and FcεRI. See Gross etal. (1992), Stancovski et al. (1993), Moritz et al. (1994), Hwu et al.(1995), Weijtens et al. (1996), and Hekele et al. (1996) for disclosuresof cTCR's using these alternative transmembrane and intracellulardomains. In a preferred embodiment, the human CD3 ζ intracellular domainwas taken for activation.

The antigen-specific extracellular domain and the intracellularsignaling-domain may be linked by a transmembrane domain, such as thehuman IgG₄Fc hinge and Fc regions. Alternatives include the human CD4transmembrane domain, the human CD28 transmembrane domain, thetransmembrane human CD3ζ domain, or a cysteine mutated human CD3ζdomain, or other transmembrane domains from other human transmembranesignaling proteins, such as CD16 and CD8 and erythropoietin receptor.

In some embodiments, the CAR nucleic acid comprises a sequence encodingother costimulatory receptors, such as a transmembrane domain and amodified CD28 intracellular signaling domain. Other costimulatoryreceptors include, but are not limited to one or more of CD28, CD27,OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to a primary signalinitiated by CD3 ζ, an additional signal provided by a humancostimulatory receptor inserted in a human CAR is important for fullactivation of T cells and could help improve in vivo persistence and thetherapeutic success of the adoptive immunotherapy.

In particular embodiments, the invention concerns isolated nucleic acidsegments and expression cassettes incorporating DNA sequences thatencode the CAR. Vectors of the present invention are designed,primarily, to deliver desired genes to immune cells, preferably T cellsunder the control of regulated eukaryotic promoters, for example, MNDU3promoter, CMV promoter, EFlalpha promoter, or Ubiquitin promoter. Also,the vectors may contain a selectable marker, if for no other reason, tofacilitate their manipulation in vitro. In other embodiments, the CARcan be expressed from mRNA in vitro transcribed from a DNA template.

Chimeric antigen receptor molecules are recombinant and aredistinguished by their ability to both bind antigen and transduceactivation signals via immunoreceptor activation motifs (ITAM's) presentin their cytoplasmic tails. Receptor constructs utilizing anantigen-binding moiety (for example, generated from single chainantibodies (scFv)) afford the additional advantage of being “universal”in that they bind native antigen on the target cell surface in anHLA-independent fashion. For example, several laboratories have reportedon scFv constructs fused to sequences coding for the intracellularportion of the CD3 complex's zeta chain (ζ), the Fc receptor gammachain, and sky tyrosine kinase (Eshhar et al., 1993; Fitzer-Attas etal., 1998). Re-directed T cell effector mechanisms including tumorrecognition and lysis by CTL have been documented in several murine andhuman antigen-scFv: ζ systems (Eshhar, 1997; Altenschmidt et al., 1997;Brocker et al., 1998).

To date non-human antigen binding regions are typically used inconstructing a chimeric antigen receptor. A potential problem with usingnon-human antigen binding regions, such as murine monoclonal antibodies,is the lack of human effector functionality and inability to penetrateinto tumor masses. In other words, such antibodies may be unable tomediate complement-dependent lysis or lyse human target cells throughantibody-dependent cellular toxicity or Fc-receptor mediatedphagocytosis to destroy cells expressing CAR. Furthermore, non-humanmonoclonal antibodies can be recognized by the human host as a foreignprotein, and therefore, repeated injections of such foreign antibodiescan lead to the induction of immune responses leading to harmfulhypersensitivity reactions. For murine-based monoclonal antibodies, thisis often referred to as a Human Anti-Mouse Antibody (HAMA) response.Therefore, the use of human antibodies is more preferred because they donot elicit as strong a HAMA response as murine antibodies. Similarly,the use of human sequences in the CAR can avoid immune-mediatedrecognition and therefore elimination by endogenous T cells that residein the recipient and recognize processed antigen in the context of HLA.

In some embodiments, the chimeric antigen receptor comprises: a) anintracellular signaling domain, b) a transmembrane domain, and c) anextracellular domain comprising an antigen binding region.

In specific embodiments, intracellular receptor signaling domains in theCAR include those of the T cell antigen receptor complex, such as thezeta chain of CD3, also Fcγ RIII costimulatory signaling domains, CD28,CD27, DAP10, CD137, OX40, CD2, alone or in a series with CD3zeta, forexample. In specific embodiments, the intracellular domain (which may bereferred to as the cytoplasmic domain) comprises part or all of one ormore of TCR zeta chain, CD28, CD27, OX40/CD134, 4-1BB/CD137, FcεRIγ,ICOS/CD278, IL-2Rbeta/CD122, IL-2Ralpha/CD132, DAP10, DAP12, and CD40.In some embodiments, one employs any part of the endogenous T cellreceptor complex in the intracellular domain. One or multiplecytoplasmic domains may be employed, as so-called third generation CARshave at least two or three signaling domains fused together for additiveor synergistic effect, for example.

In certain embodiments of the chimeric antigen receptor, theantigen-specific portion of the receptor (which may be referred to as anextracellular domain comprising an antigen binding region) comprises atumor associated antigen or a pathogen-specific antigen binding domainincluding carbohydrate antigen recognized by pattern-recognitionreceptors, such as Dectin-1. A tumor associated antigen may be of anykind so long as it is expressed on the cell surface of tumor cells.Exemplary embodiments of tumor associated antigens include CD19, CD20,carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR,c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associatedantigen, mutated p53, mutated ras, and so forth. In certain embodiments,the CAR can be co-expressed with a membrane-bound cytokine to improvepersistence when there is a low amount of tumor-associated antigen. Forexample, CAR can be co-expressed with membrane-bound IL-15.

In certain embodiments intracellular tumor associated antigens may betargeted, such as HA-1, survivin, WT1, and p53. This can be achieved bya CAR expressed on a universal T cell that recognizes the processedpeptide described from the intracellular tumor associated antigen in thecontext of HLA. In addition, the universal T cell may be geneticallymodified to express a T-cell receptor pairing that recognizes theintracellular processed tumor associated antigen in the context of HLA.

The pathogen may be of any kind, but in specific embodiments thepathogen is a fungus, bacteria, or virus, for example. Exemplary viralpathogens include those of the families of Adenoviridae, EpsteinBarrvirus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV),JC virus, BK virus, HSV, HHV family of viruses, Picornaviridae,Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae,Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus,Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses causesmallpox, influenza, mumps, measles, chickenpox, ebola, and rubella.Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus,Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenicbacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter,Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella,Chlamydia, Spirochetes, and Salmonella. In one embodiment the pathogenreceptor Dectin-1 can be used to generate a CAR that recognizes thecarbohydrate structure on the cell wall of fungi. T cells geneticallymodified to express the CAR based on the specificity of Dectin-1 canrecognize Aspergillus and target hyphal growth. In another embodiment,CARs can be made based on an antibody recognizing viral determinants(e.g., the glycoproteins from CMV and Ebola) to interrupt viralinfections and pathology.

In some embodiments, the pathogenic antigen is an Aspergilluscarbohydrate antigen for which the extracellular domain in the CARrecognizes patterns of carbohydrates of the fungal cell wall, such asvia Dectin-1.

A chimeric immunoreceptor according to the present invention can beproduced by any means known in the art, though preferably it is producedusing recombinant DNA techniques. A nucleic acid sequence encoding theseveral regions of the chimeric receptor can be prepared and assembledinto a complete coding sequence by standard techniques of molecularcloning (genomic library screening, PCR, primer-assisted ligation, scFvlibraries from yeast and bacteria, site-directed mutagenesis, etc.). Theresulting coding region can be inserted into an expression vector andused to transform a suitable expression host allogeneic T-cell line.

As used herein, a nucleic acid construct or nucleic acid sequence orpolynucleotide is intended to mean a DNA molecule that can betransformed or introduced into a T cell and be transcribed andtranslated to produce a product (e.g., a chimeric antigen receptor).

In an exemplary nucleic acid construct (polynucleotide) employed in thepresent invention, the promoter is operably linked to the nucleic acidsequence encoding the chimeric receptor of the present invention, i.e.,they are positioned so as to promote transcription of the messenger RNAfrom the DNA encoding the chimeric receptor. The promoter can be ofgenomic origin or synthetically generated. A variety of promoters foruse in T cells are well-known in the art (e.g., the CD4 promoterdisclosed by Marodon et al. (2003)). The promoter can be constitutive orinducible, where induction is associated with the specific cell type ora specific level of maturation, for example. Alternatively, a number ofwell-known viral promoters are also suitable. Promoters of interestinclude the β-actin promoter, SV40 early and late promoters,immunoglobulin promoter, human cytomegalovirus promoter, retroviruspromoter, and the Friend spleen focus-forming virus promoter. Thepromoters may or may not be associated with enhancers, wherein theenhancers may be naturally associated with the particular promoter orassociated with a different promoter.

The sequence of the open reading frame encoding the chimeric receptorcan be obtained from a genomic DNA source, a cDNA source, or can besynthesized (e.g., via PCR), or combinations thereof. Depending upon thesize of the genomic DNA and the number of introns, it may be desirableto use cDNA or a combination thereof as it is found that intronsstabilize the mRNA or provide T cell-specific expression (Barthel andGoldfeld, 2003). Also, it may be further advantageous to use endogenousor exogenous non-coding regions to stabilize the mRNA.

For expression of a chimeric antigen receptor of the present invention,the naturally occurring or endogenous transcriptional initiation regionof the nucleic acid sequence encoding N-terminal components of thechimeric receptor can be used to generate the chimeric receptor in thetarget host. Alternatively, an exogenous transcriptional initiationregion can be used that allows for constitutive or inducible expression,wherein expression can be controlled depending upon the target host, thelevel of expression desired, the nature of the target host, and thelike.

Likewise, a signal sequence directing the chimeric receptor to thesurface membrane can be the endogenous signal sequence of N-terminalcomponent of the chimeric receptor. Optionally, in some instances, itmay be desirable to exchange this sequence for a different signalsequence. However, the signal sequence selected should be compatiblewith the secretory pathway of T cells so that the chimeric receptor ispresented on the surface of the T cell.

Similarly, a termination region may be provided by the naturallyoccurring or endogenous transcriptional termination region of thenucleic acid sequence encoding the C-terminal component of the chimericreceptor. Alternatively, the termination region may be derived from adifferent source. For the most part, the source of the terminationregion is generally not considered to be critical to the expression of arecombinant protein and a wide variety of termination regions can beemployed without adversely affecting expression.

As will be appreciated by one of skill in the art that, in someinstances, a few amino acids at the ends of the antigen binding domainin the CAR can be deleted, usually not more than 10, more usually notmore than 5 residues, for example. Also, it may be desirable tointroduce a small number of amino acids at the borders, usually not morethan 10, more usually not more than 5 residues. The deletion orinsertion of amino acids may be as a result of the needs of theconstruction, providing for convenient restriction sites, ease ofmanipulation, improvement in levels of expression, or the like. Inaddition, the substitute of one or more amino acids with a differentamino acid can occur for similar reasons, usually not substituting morethan about five amino acids in any one domain.

The chimeric construct that encodes the chimeric receptor according tothe invention can be prepared in conventional ways. Because, for themost part, natural sequences may be employed, the natural genes may beisolated and manipulated, as appropriate, so as to allow for the properjoining of the various components. Thus, the nucleic acid sequencesencoding for the N-terminal and C-terminal proteins of the chimericreceptor can be isolated by employing the polymerase chain reaction(PCR), using appropriate primers that result in deletion of theundesired portions of the gene. Alternatively, restriction digests ofcloned genes can be used to generate the chimeric construct. In eithercase, the sequences can be selected to provide for restriction sitesthat are blunt-ended, or have complementary overlaps.

The various manipulations for preparing the chimeric construct can becarried out in vitro and in particular embodiments the chimericconstruct is introduced into vectors for cloning and expression in anappropriate host using standard transformation or transfection methods.Thus, after each manipulation, the resulting construct from joining ofthe DNA sequences is cloned, the vector isolated, and the sequencescreened to ensure that the sequence encodes the desired chimericreceptor. The sequence can be screened by restriction analysis,sequencing, or the like.

The chimeric constructs of the present invention find application insubjects having or suspected of having cancer by reducing the size of atumor or preventing the growth or re-growth of a tumor in thesesubjects. Accordingly, the present invention further relates to a methodfor reducing growth or preventing tumor formation in a subject byintroducing a chimeric construct of the present invention into anisolated T cell of the subject and reintroducing into the subject thetransformed T cell, thereby effecting anti-tumor responses to reduce oreliminate tumors in the subject. Suitable T cells that can be usedinclude cytotoxic lymphocytes (CTL) or any cell having a T cell receptorin need of disruption. As is well-known to one of skill in the art,various methods are readily available for isolating these cells from asubject. For example, using cell surface marker expression or usingcommercially available kits (e.g., ISOCELL™ from Pierce, Rockford,Ill.).

It is contemplated that the chimeric construct can be introduced intothe subject's own T cells as naked DNA or in a suitable vector. Methodsof stably transfecting T cells by electroporation using naked DNA areknown in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNAgenerally refers to the DNA encoding a chimeric receptor of the presentinvention contained in a plasmid expression vector in proper orientationfor expression. Advantageously, the use of naked DNA reduces the timerequired to produce T cells expressing the chimeric receptor of thepresent invention.

Alternatively, a viral vector (e.g., a retroviral vector, adenoviralvector, adeno-associated viral vector, or lentiviral vector) can be usedto introduce the chimeric construct into T cells. Suitable vectors foruse in accordance with the method of the present invention arenon-replicating in the subject's T cells. A large number of vectors areknown that are based on viruses, where the copy number of the virusmaintained in the cell is low enough to maintain the viability of thecell. Illustrative vectors include the pFB-neo vectors (STRATAGENE®)disclosed herein as well as vectors based on HIV, SV40, EBV, HSV, orBPV.

Once it is established that the transfected or transduced T cell iscapable of expressing the chimeric receptor as a surface membraneprotein with the desired regulation and at a desired level, it can bedetermined whether the chimeric receptor is functional in the host cellto provide for the desired signal induction. Subsequently, thetransduced T cells are reintroduced or administered to the subject toactivate anti-tumor responses in the subject. To facilitateadministration, the transduced T cells according to the invention can bemade into a pharmaceutical composition or made into an implantappropriate for administration in vivo, with appropriate carriers ordiluents, which further can be pharmaceutically acceptable. The means ofmaking such a composition or an implant have been described in the art(see, for instance, Remington's Pharmaceutical Sciences, 16th Ed., Mack,ed. (1980)). Where appropriate, the transduced T cells can be formulatedinto a preparation in semisolid or liquid form, such as a capsule,solution, injection, inhalant, or aerosol, in the usual ways for theirrespective route of administration. Means known in the art can beutilized to prevent or minimize release and absorption of thecomposition until it reaches the target tissue or organ, or to ensuretimed-release of the composition. Desirably, however, a pharmaceuticallyacceptable form is employed that does not ineffectuate the cellsexpressing the chimeric receptor. Thus, desirably the transduced T cellscan be made into a pharmaceutical composition containing a balanced saltsolution, preferably Hanks' balanced salt solution, or normal saline.

III. METHODS AND COMPOSITIONS RELATED TO THE EMBODIMENTS

In certain aspects, the invention includes a method of making and/orexpanding the antigen-specific redirected T cells that comprisestransfecting T cells with an expression vector containing a DNAconstruct encoding the hCAR, then, optionally, stimulating the cellswith antigen positive cells, recombinant antigen, or an antibody to thereceptor to cause the cells to proliferate.

In another aspect, a method is provided of stably transfecting andre-directing T cells by electroporation, or other non-viral genetransfer (such as, but not limited to sonoporation) using naked DNA.Most investigators have used viral vectors to carry heterologous genesinto T cells. By using naked DNA, the time required to produceredirected T cells can be reduced. “Naked DNA” means DNA encoding achimeric T-cell receptor (cTCR) contained in an expression cassette orvector in proper orientation for expression. The electroporation methodof this invention produces stable transfectants that express and carryon their surfaces the chimeric TCR (cTCR).

“Chimeric TCR” means a receptor that is expressed by T cells and thatcomprises intracellular signaling, transmembrane, and extracellulardomains, where the extracellular domain is capable of specificallybinding in an MHC unrestricted manner an antigen that is not normallybound by a T-cell receptor in that manner Stimulation of the T cells bythe antigen under proper conditions results in proliferation (expansion)of the cells and/or production of IL-2. The exemplary CD19- and HERV-Kspecific chimeric receptors of the instant application are examples of achimeric TCR. However, the method is applicable to transfection withchimeric TCRs that are specific for other target antigens, such aschimeric TCRs that are specific for HER2/Neu (Stancovski et al., 1993),ERBB2 (Moritz et al., 1994), folate binding protein (Hwu et al., 1995),renal cell carcinoma (Weitjens et al., 1996), and HIV-1 envelopeglycoproteins gp120 and gp41 (Roberts et al., 1994). Other cell-surfacetarget antigens include, but are not limited to, CD20, carcinoembryonicantigen, mesothelin, ROR1, c-Met, CD56, GD2, GD3, alphafetoprotein,CD23, CD30, CD123, IL-11Ralpha, kappa chain, lambda chain, CD70, CA-125,MUC-1, EGFR and variants, epithelial tumor antigen, and so forth.

In certain aspects, the T cells are primary human T cells, such as Tcells derived from human peripheral blood mononuclear cells (PBMC), PBMCcollected after stimulation with G-CSF, bone marrow, or umbilical cordblood. Conditions include the use of mRNA and DNA and electroporation.Following transfection the cells may be immediately infused or may bestored. In certain aspects, following transfection, the cells may bepropagated for days, weeks, or months ex vivo as a bulk populationwithin about 1, 2, 3, 4, 5 days or more following gene transfer intocells. In a further aspect, following transfection, the transfectantsare cloned and a clone demonstrating presence of a single integrated orepisomally maintained expression cassette or plasmid, and expression ofthe chimeric receptor is expanded ex vivo. The clone selected forexpansion demonstrates the capacity to specifically recognize and lyseCD19 expressing target cells. The recombinant T cells may be expanded bystimulation with IL-2, or other cytokines that bind the commongamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others). Therecombinant T cells may be expanded by stimulation with artificialantigen presenting cells. The recombinant T cells may be expanded onartificial antigen presenting cell or with an antibody, such as OKT3,which cross links CD3 on the T cell surface. Subsets of the recombinantT cells may be deleted on artificial antigen presenting cell or with anantibody, such as Campath, which binds CD52 on the T cell surface. In afurther aspect, the genetically modified cells may be cryopreserved.

T-cell propagation (survival) after infusion may be assessed by: (i)q-PCR using primers specific for the CAR; (ii) flow cytometry using anantibody specific for the CAR; and/or (iii) soluble TAA.

Embodiments of the invention also concern the targeting of a B-cellmalignancy or disorder including B cells, with the cell-surface epitopebeing CD19-specific using a redirected immune T cell. Malignant B cellsare an excellent target for redirected T cells, as B cells can serve asimmunostimulatory antigen-presenting cells for T cells. Preclinicalstudies that support the anti-tumor activity of adoptive therapy withdonor-derived CD19-specific T-cells bearing a human or humanized CARinclude (i) redirected killing of CD19⁺ targets, (ii) redirectedsecretion/expression of cytokines after incubation with CD19⁺targets/stimulator cells, and (iii) sustained proliferation afterincubation with CD19⁺ targets/stimulator cells.

In certain embodiments of the invention, the CAR cells are delivered toan individual in need thereof, such as an individual that has cancer oran infection. The cells then enhance the individual's immune system toattack the respective cancer or pathogenic cells. In some cases, theindividual is provided with one or more doses of the antigen-specificCAR T-cells. In cases where the individual is provided with two or moredoses of the antigen-specific CAR T-cells, the duration between theadministrations should be sufficient to allow time for propagation inthe individual, and in specific embodiments the duration between dosesis 1, 2, 3, 4, 5, 6, 7, or more days.

The source of the allogeneic T cells that are modified to include both achimeric antigen receptor and that lack functional TCR may be of anykind, but in specific embodiments the cells are obtained from a bank ofumbilical cord blood, peripheral blood, human embryonic stem cells, orinduced pluripotent stem cells, for example. Suitable doses for atherapeutic effect would be at least 10⁵ or between about 10⁵ and about10¹⁰ cells per dose, for example, preferably in a series of dosingcycles. An exemplary dosing regimen consists of four one-week dosingcycles of escalating doses, starting at least at about 10⁵ cells on Day0, for example increasing incrementally up to a target dose of about10¹⁰ cells within several weeks of initiating an intra-patient doseescalation scheme. Suitable modes of administration include intravenous,subcutaneous, intracavitary (for example by reservoir-access device),intraperitoneal, and direct injection into a tumor mass.

A pharmaceutical composition of the present invention can be used aloneor in combination with other well-established agents useful for treatingcancer. Whether delivered alone or in combination with other agents, thepharmaceutical composition of the present invention can be delivered viavarious routes and to various sites in a mammalian, particularly human,body to achieve a particular effect. One skilled in the art willrecognize that, although more than one route can be used foradministration, a particular route can provide a more immediate and moreeffective reaction than another route. For example, intradermal deliverymay be advantageously used over inhalation for the treatment ofmelanoma. Local or systemic delivery can be accomplished byadministration comprising application or instillation of the formulationinto body cavities, inhalation or insufflation of an aerosol, or byparenteral introduction, comprising intramuscular, intravenous,intraportal, intrahepatic, peritoneal, subcutaneous, or intradermaladministration.

A composition of the present invention can be provided in unit dosageform wherein each dosage unit, e.g., an injection, contains apredetermined amount of the composition, alone or in appropriatecombination with other active agents. The term unit dosage form as usedherein refers to physically discrete units suitable as unitary dosagesfor human and animal subjects, each unit containing a predeterminedquantity of the composition of the present invention, alone or incombination with other active agents, calculated in an amount sufficientto produce the desired effect, in association with a pharmaceuticallyacceptable diluent, carrier, or vehicle, where appropriate. Thespecifications for the novel unit dosage forms of the present inventiondepend on the particular pharmacodynamics associated with thepharmaceutical composition in the particular subject.

Desirably an effective amount or sufficient number of the isolatedtransduced T cells is present in the composition and introduced into thesubject such that long-term, specific, anti-tumor responses areestablished to reduce the size of a tumor or eliminate tumor growth orregrowth than would otherwise result in the absence of such treatment.Desirably, the amount of transduced T cells reintroduced into thesubject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%,or 100% decrease in tumor size when compared to otherwise sameconditions wherein the transduced T cells are not present.

Accordingly, the amount of transduced T cells administered should takeinto account the route of administration and should be such that asufficient number of the transduced T cells will be introduced so as toachieve the desired therapeutic response. Furthermore, the amounts ofeach active agent included in the compositions described herein (e.g.,the amount per each cell to be contacted or the amount per certain bodyweight) can vary in different applications. In general, theconcentration of transduced T cells desirably should be sufficient toprovide in the subject being treated at least from about 1×10⁶ to about1×10⁹ transduced T cells, even more desirably, from about 1×10⁷ to about5×10⁸ transduced T cells, although any suitable amount can be utilizedeither above, e.g., greater than 5×10⁸ cells, or below, e.g., less than1×10⁷ cells. The dosing schedule can be based on well-establishedcell-based therapies (see, e.g., Topalian and Rosenberg, 1987; U.S. Pat.No. 4,690,915), or an alternate continuous infusion strategy can beemployed.

These values provide general guidance of the range of transduced T cellsto be utilized by the practitioner upon optimizing the method of thepresent invention for practice of the invention. The recitation hereinof such ranges by no means precludes the use of a higher or lower amountof a component, as might be warranted in a particular application. Forexample, the actual dose and schedule can vary depending on whether thecompositions are administered in combination with other pharmaceuticalcompositions, or depending on interindividual differences inpharmacokinetics, drug disposition, and metabolism. One skilled in theart readily can make any necessary adjustments in accordance with theexigencies of the particular situation.

IV. EXEMPLARY HUMAN CD19-SPECIFIC CHIMERIC ANTIGEN RECEPTOR T CELLS

CD19, a cell surface glycoprotein of the immunoglobulin superfamily, isa potentially attractive target for antibody therapy of Bcell-associated malignancies. This antigen is absent from hematopoieticstem cells, and in healthy individuals its presence is exclusivelyrestricted to the B-lineage and possibly some follicular dendritic cells(Scheuermann et al., 1995). In fact, it is present on B cells from theearliest recognizable B-lineage cells during development to B-cellblasts but is lost on maturation to plasma cells. Furthermore, CD19 isnot shed from the cell surface and rarely lost during neoplastictransformation (Scheuermann et al., 1995). The protein is expressed onmost malignant B-lineage cells, including cells from patients withchronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), andacute lymphoblastic leukemia (ALL) (Uckun et al., 1988). CD19 primarilyacts as a B cell co-receptor in conjunction with CD21 and CD81. Uponactivation, the cytoplasmic tail of CD19 becomes phosphorylated, whichleads to binding by Src-family kinases and recruitment of PI-3 kinase.

In one aspect compositions and methods of the embodiments concern humanCD19-specific chimeric T cell receptor (or chimeric antigen receptor,CAR) polypeptide (designated hCD19CAR) comprising an intracellularsignaling domain, a transmembrane domain, and an extracellular domain,the extracellular domain comprising a human CD19 binding region. Inanother aspect, the CD19 binding region is a F(ab′)2, Fab′, Fab, Fv, orscFv. The binding region may comprise an amino acid sequence that is atleast, at most, or about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to the wild-type amino acid sequence. Theintracellular domain may comprise an intracellular signaling domain ofhuman CD3ζ and may further comprise human CD28 intracellular segment. Incertain aspects the transmembrane domain is a CD28 transmembrane domain.

In a further aspect compositions of the invention include a nucleic acidencoding the polypeptide described above. In certain aspects the nucleicacid sequence is optimized for human codon usage.

In still a further aspect compositions of the invention include cellsexpressing the polypeptide described herein. The T cell may comprise anexpression cassette encoding hCD19CAR polypeptide. The expressioncassette can be comprised in a non-viral vector, such as a transposon,or a human transposon, or recombinant variant thereof. The expressioncassette can be comprised in a viral vector or recombinant variantthereof. The expression cassette can be genomically integrated orepisomally maintained or expressed from mRNA.

In yet a further aspect the invention includes a method of making a Tcell expressing a human CD19-specific CAR comprising introducing anexpression cassette into the cell, wherein the expression cassetteencodes a polypeptide comprising a human extracellular CD19 bindingdomain, a transmembrane domain, and one or more intracellular signalingdomain(s). The method may further comprise stimulating the cells withCD19⁺ cells, recombinant CD19, or an antibody to the receptor to causethe cells to proliferate, kill, and/or make cytokines; for example, thecells may be stimulated to proliferate or expand with CD19⁺ artificialantigen presenting cells.

In certain aspects the invention includes methods of treating a humandisease condition associated with a cell expressing endogenous CD19comprising infusing a patient with an amount of a recombinant cellexpressing a human CD19-specific CAR sufficient to treat the condition,wherein the human CD19-specific CAR comprises a human CD19 extracellularbinding domain, a transmembrane domain, and an intracellular signalingdomain. The condition can be lymphoma, leukemia, Non-Hodgkin's lymphoma,acute lymphoblastic leukemia, chronic lymphoblastic leukemia, chroniclymphocytic leukemia, or B cell-associated autoimmune diseases, forexample.

The invention relates to the generation of a human CD19-specificchimeric antigen receptor (hCD19RCD28 or hCAR). In certain aspectsrecombinant cells expressing hCAR have improved in vivo persistence andanti-tumor efficacy. The human hCAR has a reduced immunogenicitycompared to murine hCAR, which comprises a scFv segment derived from amurine CD19-specific monoclonal antibody (mAb). Anti-tumor effects canbe augmented by genetically modified cells, rendered specific for CD19.Typically, T cell specificity is achieved by electrotransfer of anexpression cassette encoding hCAR.

The hCAR may be a chimeric receptor comprising one or more activationendodomain(s), such as a CD3-ζ-derived activation domain. AdditionalT-cell activation motifs include, but are not limited to, CD28, CD27,OX-40, DAP10, and 4-1BB. In certain aspects the activation domain canalso include a CD28 transmembrane and/or activation domain. In a furtheraspect the hCAR encoding region and/or expression cassette codonoptimized for expression in human cells and subjects, e.g., in oneembodiment the scFv region obtained from VH and VL sequences of aCD19-specific human antibodies are incorporated into the CD19 bindingsegment of the hCAR (for example see U.S. Pat. No. 7,109,304, which isincorporated herein by reference in its entirety). In anotherembodiment, the hCAR expression cassette is episomally maintained orintegrated into the genome of the recombinant cell. In certain aspectsthe expression cassette is comprised in a nucleic acid capable ofintegration by using an integrase mechanism, a viral vector, such as aretroviral vector, or a nonviral vector, such as a transposon mechanism.In a further embodiment the expression cassette is included in atransposon based nucleic acid. In a particular embodiment, theexpression cassette is part of a two component Sleeping Beauty (SB) orpiggyBac system that utilizes a transposon and transposase for enhancednon-viral gene transfer.

Recombinant hCAR expressing cells can be numerically expanded toclinically-meaningful numbers. One example of such expansion usesartificial antigen presenting cells (aAPC). Recombinant hCAR expressingcells can be verified and identified by flow cytometry and western blotanalyses. Recombinant hCAR expressing T cells, expressing aCD19-specific CAR can recognize and kill CD19 expressing target cells.In a further aspect, hCAR can be expressed into Universal cells that canbe infused across transplantation barriers to help preventimmunogenicity. The hCAR can be used along with human genes for imaging(such as by positron emission tomography, PET) and conditional ablationof T cells, in the event of cytotoxicity. The recombinant cells of theinvention can be used in CD19-specific cellular therapies.

V. EXEMPLARY HERV-TARGETING CHIMERIC ANTIGEN RECEPTOR T CELLS

During the human genome project, a set of ancient retrovirus named humanendogenous retrovirus (HERVs) was discovered to be stably integratedinto the human genome forming 8.5% of the total human genome. Among theHERVs, HERV-K was found as an oncogenic allelic variant involved inmelanoma, breast cancer, ovarian cancer, teratocarcinoma and prostatecancer along with various autoimmune diseases like multiple sclerosisand rheumatoid arthritis (Buscher et al., 2005; Dreyfus, 2011; Seifarthet al., 1995). The oncogenic potential of HERV-K is contributed by theenvelope (env) and GAG protein (Rec). Recent studies have shown that theexpression of the HERV-K env protein exclusively on tumor cell surfaceand not on normal skin cells (Wang-Johanning et al., 2008; Li et al.,2010). The expression of HERV-K env protein was found to increase withmore aggressive and metastatic type III and type IV melanoma than lessaggressive and localized type I melanoma (Buscher et al., 2005; Hahn etal., 2008; Serafino et al., 2009). This selective expression of HERV-Kenv protein on melanoma cells can be harnessed as a treatment strategyfor patients with refractory or metastatic melanoma. Patients withmetastatic melanoma have a poor prognosis due to resistance toconventional therapies such as chemotherapy, radiation and surgery(Bhatia et al., 2009). Thus, new targeted treatment strategies areneeded to improve therapeutic outcome.

To generate T-cell therapy for melanoma, the inventors targeted a TAAderived from HERV, whose genome stably integrated into humans millionsof years ago. To target HERK-K, T cells were engineered to express a CARspecific for the env protein by replacing the antigen-binding exodomainof CD19-specific CAR with the single chain antibody (scFv) sequence ofan anti-HERV-K env-specific monoclonal antibody. This new CAR was clonedas a transposon into the SB system. DNA plasmids coding for the HERV-Kenv-specific CAR and SB transposase were electro-transferred intoprimary human T cells and genetically modified CAR⁺ T cells wereselectively propagated on irradiated artificial antigen presenting cells(aAPC) expressing HERV-K env and desired T-cell co-stimulatorymolecules. After co-culture on γ-irradiated aAPC, 95% of CD3⁺ T cellsexpressed the CAR and these CAR⁺ T cells were able to specifically killHERV-K env⁺, but not HERV-K env⁻, tumor targets in vitro in contrast toCAR⁺ T cells. Specificity of these CAR⁺ T cells was proven by expressingthe HERV-K env protein in antigen negative EL4 mouse cells which werepreferentially killed compared to HERV-K env EL4 parental cells andHERV-K knockdown by shRNA (specific targeting for HERV-K env RNA) onA888-mel cells resulted in reduced killing compared to parental. TheCAR⁺ T cells were also successful in reducing tumor growth andmetastasis of A375-super metastatic (SM) tumor cells from lungs to liverin vivo. The mice with tumor receiving the CAR⁺ T cells lived longer andappeared healthier than the tumor alone mice group. Thus, T cellstargeting an active retrovirus can be used as an immunotherapy formelanoma, using an approach that has translational appeal for clinicaltrials.

Clonal evolution of melanoma cells can render the TCR therapyineffective due to its dependency on MHC complex for tumor recognition.A CAR can overcome this clonal evolution of melanoma cells by mediatedcell death in a MHN independent manner, unlike TCR-based T-cell therapy.The HERV-K antigen is expressed only on the tumor cell surface and noton normal cells. Therefore, HERV-K-specific CAR⁺ T cells canspecifically target and abolish tumor cells without any adverse sideeffects. They may be infused to treat HERV-K⁺ malignancies at manystages along the cancer spectrum, from minimal residual disease, to bulktumors, to disease that is refractory to conventional therapies.

HERV-K is expressed on many tumor types, including, but not limited to,melanoma (Muster et al., 2003; Buscher et al., 2005; Li et al., 2010;Reiche et al., 2010; Serafino et al., 2009), breast cancer (Patience etal., 1996; Wang-Johanning et al., 2003; Seifarth et al., 1995), ovariancancer (Wang-Johanning et al., 2007), lymphoma (Contreras-Galindo etal., 2008), and teratocarcinoma (Bieda et al., 2001; Lower et al.,1993). Furthermore, infected cells, including those infected by HIV(Jones et al., 2012), also express HERV-K. This provides an attractiveopportunity that one CAR design targeting HERV-K may be used to treat avariety of cancers and infections.

VI. EXEMPLARY MEMBRANE-BOUND IL-15 CO-EXPRESSING CHIMERIC ANTIGENRECEPTOR T CELLS FOR TARGETING MINIMALLY RESIDUAL DISEASE

Chemotherapeutic treatment of adult and pediatric B-lineage acutelymphoblastic leukemia (B-ALL) have disease relapse rates of 65% and20%, respectively, due to drug-resistant residual disease. The highincidence of B-ALL relapse, especially in poor prognostic groups, hasprompted the use of immune-based therapies using allogeneichematopoietic stem cell transplantation (HSCT). This therapy isdependent on alloreactive cells present in the donor graft for theeradication of remaining leukemic cells, or minimal residual disease, toimprove disease-free survival. Donor lymphocyte infusions have been usedto enhance the ability of engrafted T cells to target residual B-ALLafter allogeneic HSCT, but this treatment approach for such patientsachieves less than a 10% remission rate and is associated with a highdegree of morbidity and mortality from the frequency and severity ofgraft-versus-host disease (GVHD). With relapse a common and lethalproblem in these refractory malignancies, adoptive therapy usingperipheral blood mononuclear cells (PBMC)-derived T cells after HSCT maybe used to increase the anti-tumor effect, or graft-versus-leukemia(GVL) effect, by retargeting the specificity of donor T cells to atumor-associated antigen (TAA).

Numerous formulations of CARs specific for target antigens have beendeveloped, with CD19-specific CAR targeting the CD19 antigen on the cellsurface of B-ALL. Observing long-term persistence of CAR⁺ cells andachieving durable responses in patients across different clinicalprotocols remains a critical issue hampering the therapeutic efficacy ofCAR-based therapies.

Currently, CAR-modified T cells are reliant on obtaining survivalsignaling through the CAR which occurs only upon encounter with thetumor antigen. In clinical situations where these CAR-modified T cellsare infused into patients with bulky disease, there is ample tumorantigen present to provide sufficient activation and survival signalingvia the CAR. However, patients with relapsed B-ALL are often conditionedwith myeloablative chemotherapy followed by HSCT and present withminimal residual disease (MRD). In this case, patients have a low tumorload and the minute level of TAA severely restricts the CAR-mediatedsignaling necessary for supporting the infused T cells consequentlycompromising therapeutic potential. An alternate CAR-independent meansfor improving T cell persistence would be anticipated to improve theengraftment of CAR-modified T cells.

Cytokines in the common gamma chain receptor family (γC) are importantcostimulatory molecules for T cells that are critical to lymphoidfunction, survival, and proliferation. IL-15 possesses severalattributes that are desirable for adoptive therapy. IL-15 is ahomeostatic cytokine that supports the survival of long-lived memorycytotoxic T cells, promotes the eradication of established tumors viaalleviating functional suppression of tumor-resident cells, and inhibitsAICD.

IL-15 is tissue restricted and only under pathologic conditions is itobserved at any level in the serum, or systemically. Unlike other γCcytokines that are secreted into the surrounding milieu, IL-15 istrans-presented by the producing cell to T cells in the context of IL-15receptor alpha (IL-15Rα). The unique delivery mechanism of this cytokineto T cells and other responding cells: (i) is highly targeted andlocalized, (ii) increases the stability and half-life of IL-15, and(iii) yields qualitatively different signaling than is achieved bysoluble IL-15.

In one embodiment, the invention provides a method of generatingchimeric antigen receptor (CAR)-modified T cells with long-lived in vivopotential for the purpose of treating, for example, leukemia patientsexhibiting minimal residual disease (MRD). In aggregate, this methoddescribes how soluble molecules such as cytokines can be fused to thecell surface to augment therapeutic potential. The core of this methodrelies on co-modifying CAR T cells with a human cytokine mutein ofinterleukin-15 (IL-15), henceforth referred to as mIL15. The mIL15fusion protein is comprised of codon-optimized cDNA sequence of IL-15fused to the full length IL15 receptor alpha via a flexibleserine-glycine linker. This IL-15 mutein was designed in such a fashionso as to: (i) restrict the mIL15 expression to the surface of the CAR⁺ Tcells to limit diffusion of the cytokine to non-target in vivoenvironments, thereby potentially improving its safety profile asexogenous soluble cytokine administration has led to toxicities; and(ii) present IL-15 in the context of IL-15Rα to mimic physiologicallyrelevant and qualitative signaling as well as stabilization andrecycling of the IL-15/IL-15Ra complex for a longer cytokine half-life.T cells expressing mIL15 are capable of continued supportive cytokinesignaling, which is critical to their survival post-infusion. ThemIL15⁺CAR⁺ T cells generated by non-viral Sleeping Beauty System geneticmodification and subsequent ex vivo expansion on a clinically applicableplatform yielded a T cell infusion product with enhanced persistenceafter infusion in murine models with high, low, or no tumor burden.Moreover, the mIL15⁺CAR⁺ T cells also demonstrated improved anti-tumorefficacy in both the high or low tumor burden models.

The improved persistence and anti-tumor activity of mIL15⁺CAR⁺ T cellsover CAR⁺ T cells in the high tumor burden model indicates thatmIL15⁺CAR⁺ T cells may be more efficacious than CAR⁺ T cells in treatingleukemia patients with active disease where tumor burden is prevalent.Thus, mIL15⁺CAR⁺ T cells could supplant CAR⁺ T cells in adoptive therapyin the broadest of applications. The capability of mIL15⁺CAR⁺ T cells tosurvive independent of survival signaling via the CAR enables thesemodified T cells to persist post-infusion despite the lack of tumorantigen. Consequently, this is anticipated to generate the greatestimpact in therapeutic efficacy in a MRD treatment setting, especially inpatients who have had myeloablative chemotherapy and hematopoietic stemcell transplantation. These patients would receive adoptive T celltransfer with mIL15⁺CAR⁺ T cells to treat their MRD and prevent relapse.

Membrane-bound cytokines, such as mIL15, have broad implications. Inaddition to membrane-bound IL-15, other membrane-bound cytokines areenvisioned. The membrane-bound cytokines can also be extended to cellsurface expression of other molecules associated with activating andpropagating cells used for human application. These include, but are notlimited to cytokines, chemokines, and other molecules that contribute tothe activation and proliferation of cells used for human application.

Membrane-bound cytokine, such as mIL15 can be used ex vivo to preparecells for human application(s) and can be on infused cells (e.g., Tcells) used for human application. For example, membrane-bound IL-15 canbe expressed on artificial antigen presenting cells (aAPC), such asderived from K562, to stimulate T cells and NK cells (as well as othercells) for activation and/or proliferation. The population of T cellsactivated/propagated by mIL15 on aAPC includes genetically modifiedlymphocytes, but also tumor-infiltrating lymphocytes, and other immunecells. These aAPC are not infused. In contrast, mIL15 (and othermembrane-bound molecules) can be expressed on T cells, and other cells,that are infused.

Therapeutic efficacy of MRD treatment with CAR-modified T cells ishampered by a lack of persistence after adoptive transfer of the Tcells. The capability of mIL15⁺CAR⁺ T cells to survive long-term in vivoindependently of tumor antigen indicates great potential for treatingpatients with MRD. In this case, mIL15 and the persisting T cells thatit supports would address a need, as current approaches for MRD patientsare insufficient. The persistence of T cells and other lymphocytes thatare infused in patients with MRD applies beyond CAR⁺ T cells. Any immunecell that is used to treat and prevent malignancy, infection orauto-immune disease must be able to persist over the long term ifcontinued therapeutic impact is to be achieved. Thus, activating T cellsfor persistence beyond the signal derived from endogenous T-cellreceptor or an introduced immunoreceptor is important to many aspects ofadoptive immunotherapy. The expression of membrane-bound cytokine(s)thus can be used to augment the therapeutic potential and persistence ofT cells and other immune cells infused for a variety of pathologicconditions.

The inventors have generated a mutein of IL-15 that is expressed as amembrane-bound fusion protein of IL-15 and IL-15Rα (mIL15) on CAR⁺ Tcells. The mIL15 construct was co-electro-transferred with aCD19-specific CAR (on Day 0) into primary human T cells as two SleepingBeauty DNA transposon plasmids. Clinically relevant numbers ofmIL15⁺CAR⁺ T cells were generated by co-culture on CD19⁺ artificialantigen presenting cells and supplemented IL-21. Signaling through theIL-15 receptor complex in genetically modified T cells was validated byphosphorylation of STAT5 (pSTAT5) and these T cells demonstratedredirected specific lysis of CD19⁺ tumor targets equivalent to CAR⁺ Tcells. Furthermore, after antigen withdrawal, signaling generated bymIL15 increased the prevalence of T cells with a lessdifferentiated/younger phenotype that possessed memory-associatedattributes including specific cell surface markers, transcriptionfactors, and the capacity to secrete IL-2. These characteristics aredesirable traits in T cells used in adoptive transfer as they arecorrelated with T cell subsets with demonstrated capability to persistlong-term in vivo. In immunocompromised NSG mice bearing a disseminatedCD19⁺ leukemia, the mIL15⁺CAR⁺ T cells demonstrated both persistence andan anti-tumor effect whereas its CAR⁺ T cell counterpart could notmaintain significant persistence despite the presence of TAA. In apreventative mouse (NSG) model where mIL15^(+/−)CAR⁺ T cells were firstengrafted for six days followed by the introduction of a disseminatedCD19⁺ leukemia, only the mIL15⁺CAR⁺ T cells were found to persist aswell as prevent tumor engraftment. To test whether mIL15⁺CAR⁺ T cellswere capable of persistence independent of stimulation from TAA,mIL15^(+/−)CAR⁺ T cells were adoptively transferred into NSG mice withno tumor. Only mIL15⁺CAR⁺ T cells were capable of persisting in this invivo environment without exogenous cytokine support or the presence ofCD19 TAA. These data demonstrate that mIL15 can be co-expressed on CAR⁺T cells resulting in enhanced in vivo persistence without the need forTAA or exogenous cytokine support. In summary, this cytokine fusionmolecule: (i) provides stimulatory signals via pSTAT5 leading toaugmented in vivo T-cell persistence while maintaining tumor-specificfunctionality, (ii) maintains T-cell subsets that promotes a memory-likephenotype, (iii) eliminates the need and cost for clinical-grade IL-2for in vitro and in vivo T-cell expansion and persistence, and (iv)mitigates the need for clinical-grade soluble IL-15.

VII. IMMUNE SYSTEM AND IMMUNOTHERAPY

In some embodiments, a medical disorder is treated by transfer of aredirected T cell that elicits a specific immune response. In oneembodiment of the present invention, B-cell lineage malignancy ordisorder is treated by transfer of a redirected T cell that elicits aspecific immune response. Thus, a basic understanding of the immunologicresponses is necessary.

The cells of the adaptive immune system are a type of leukocyte, calleda lymphocyte. B cells and T cells are the major types of lymphocytes. Bcells and T cells are derived from the same pluripotent hematopoieticstem cells, and are indistinguishable from one another until after theyare activated. B cells play a large role in the humoral immune response,whereas T cells are intimately involved in cell-mediated immuneresponses. They can be distinguished from other lymphocyte types, suchas B cells and NK cells by the presence of a special receptor on theircell surface called the T-cell receptor (TCR). In nearly all othervertebrates, B cells and T cells are produced by stem cells in the bonemarrow. T cells travel to and develop in the thymus, from which theyderive their name. In humans, approximately 1%-2% of the lymphocyte poolrecirculates each hour to optimize the opportunities forantigen-specific lymphocytes to find their specific antigen within thesecondary lymphoid tissues.

T lymphocytes arise from hematopoietic stem cells in the bone marrow,and typically migrate to the thymus gland to mature. T cells express aunique antigen binding receptor on their membrane (T-cell receptor),which can only recognize antigen in association with majorhistocompatibility complex (MHC) molecules on the surface of othercells. There are at least two populations of T cells, known as T helpercells and T cytotoxic cells. T helper cells and T cytotoxic cells areprimarily distinguished by their display of the membrane boundglycoproteins CD4 and CD8, respectively. T helper cells secret variouslymphokines that are crucial for the activation of B cells, T cytotoxiccells, macrophages, and other cells of the immune system. In contrast, Tcytotoxic cells that recognize an antigen-MHC complex proliferate anddifferentiate into effector cell called cytotoxic T lymphocytes (CTLs).CTLs eliminate cells of the body displaying antigen, such as virusinfected cells and tumor cells, by producing substances that result incell lysis. Natural killer cells (or NK cells) are a type of cytotoxiclymphocyte that constitutes a major component of the innate immunesystem. NK cells play a major role in the rejection of tumors and cellsinfected by viruses. The cells kill by releasing small cytoplasmicgranules of proteins called perforin and granzyme that cause the targetcell to die by apoptosis.

Antigen-presenting cells, which include macrophages, B lymphocytes, anddendritic cells, are distinguished by their expression of a particularMHC molecule. APCs internalize antigen and re-express a part of thatantigen, together with the MHC molecule on their outer cell membrane.The major histocompatibility complex (MHC) is a large genetic complexwith multiple loci. The MHC loci encode two major classes of MHCmembrane molecules, referred to as class I and class II MHCs. T helperlymphocytes generally recognize antigen associated with MHC class IImolecules, and T cytotoxic lymphocytes recognize antigen associated withMHC class I molecules. In humans the MHC is referred to as the HLAcomplex and in mice the H-2 complex.

The T-cell receptor, or TCR, is a molecule found on the surface of Tlymphocytes (or T cells) that is generally responsible for recognizingantigens bound to major histocompatibility complex (MHC) molecules. Itis a heterodimer consisting of an alpha and beta chain in 95% of Tcells, while 5% of T cells have TCRs consisting of gamma and deltachains. Engagement of the TCR with antigen and MHC results in activationof its T lymphocyte through a series of biochemical events mediated byassociated enzymes, co-receptors, and specialized accessory molecules.In immunology, the CD3 antigen (CD stands for cluster ofdifferentiation) is a protein complex composed of four distinct chains(CD3-γ, CD3δ, and two times CD3ε) in mammals, that associate withmolecules known as the T-cell receptor (TCR) and the ζ-chain to generatean activation signal in T lymphocytes. The TCR, ζ-chain, and CD3molecules together comprise the TCR complex. The CD3-γ, CD3δ, and CD3εchains are highly related cell surface proteins of the immunoglobulinsuperfamily containing a single extracellular immunoglobulin domain. Thetransmembrane region of the CD3 chains is negatively charged, acharacteristic that allows these chains to associate with the positivelycharged TCR chains (TCRα and TCRβ). The intracellular tails of the CD3molecules contain a single conserved motif known as an immunoreceptortyrosine-based activation motif or ITAM for short, which is essentialfor the signaling capacity of the TCR.

CD28 is one of the molecules expressed on T cells that provideco-stimulatory signals, which are required for T cell activation. CD28is the receptor for B7.1 (CD80) and B7.2 (CD86). When activated byToll-like receptor ligands, the B7.1 expression is upregulated inantigen presenting cells (APCs). The B7.2 expression on antigenpresenting cells is constitutive. CD28 is the only B7 receptorconstitutively expressed on naive T cells. Stimulation through CD28 inaddition to the TCR can provide a potent co-stimulatory signal to Tcells for the production of various interleukins (IL-2 and IL-6 inparticular).

The strategy of isolating and expanding antigen-specific T cells as atherapeutic intervention for human disease has been validated inclinical trials (Riddell et al., 1992; Walter et al., 1995; Heslop etal., 1996).

Malignant B cells appear to be an excellent targets for redirected Tcells, as B cells can serve as immunostimulatory antigen-presentingcells for T cells (Glimcher et al., 1982). Lymphoma, by virtue of itslymph node tropism, is anatomically ideally situated for T cell-mediatedrecognition and elimination. The localization of infused T cells tolymph node in large numbers has been documented in HIV patientsreceiving infusions of HIV-specific CD8⁺ CTL clones. In these patients,evaluation of lymph node biopsy material revealed that infused clonesconstituted approximately 2%-8% of CD8⁺ cells of lymph nodes. Lymph nodehoming might be further improved by co-transfecting T cells with a cDNAconstruct encoding the L-selection molecule under a constitutivepromoter since this adhesion molecule directs circulating T cells backto lymph nodes and is down-regulated by in vitro expansion (Chao et al.,1997). The present invention may provide a method of treating a humandisease condition associated with a cell expressing endogenous CD19comprising infusing a patient with a therapeutically effective dose ofthe recombinant human CD19-specific CAR expressing cell as describedabove. The human disease condition associated with a cell expressingendogenous CD19 may be selected from the group consisting of lymphoma,leukemia, non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chroniclymphoblastic leukemia, chronic lymphocytic leukemia, and Bcell-associated autoimmune diseases.

Leukemia is a cancer of the blood or bone marrow and is characterized byan abnormal proliferation (production by multiplication) of blood cells,usually white blood cells (leukocytes). It is part of the broad group ofdiseases called hematological neoplasms. Leukemia is a broad termcovering a spectrum of diseases. Leukemia is clinically andpathologically split into its acute and chronic forms.

Acute leukemia is characterized by the rapid proliferation of immatureblood cells. This crowding makes the bone marrow unable to producehealthy blood cells. Acute forms of leukemia can occur in children andyoung adults. In fact, it is a more common cause of death for childrenin the U.S. than any other type of malignant disease. Immediatetreatment is required in acute leukemia due to the rapid progression andaccumulation of the malignant cells, which then spill over into thebloodstream and spread to other organs of the body. Central nervoussystem (CNS) involvement is uncommon, although the disease canoccasionally cause cranial nerve palsies. Chronic leukemia isdistinguished by the excessive build up of relatively mature, but stillabnormal, blood cells. Typically taking months to years to progress, thecells are produced at a much higher rate than normal cells, resulting inmany abnormal white blood cells in the blood. Chronic leukemia mostlyoccurs in older people, but can theoretically occur in any age group.Whereas acute leukemia must be treated immediately, chronic forms aresometimes monitored for some time before treatment to ensure maximumeffectiveness of therapy.

Furthermore, the diseases are classified into lymphocytic orlymphoblastic, which indicate that the cancerous change took place in atype of marrow cell that normally goes on to form lymphocytes, andmyelogenous or myeloid, which indicate that the cancerous change tookplace in a type of marrow cell that normally goes on to form red cells,some types of white cells, and platelets (see lymphoid cells vs. myeloidcells).

Acute lymphocytic leukemia (also known as acute lymphoblastic leukemia,or ALL) is the most common type of leukemia in young children. Thisdisease also affects adults, especially those aged 65 and older. Chroniclymphocytic leukemia (CLL) most often affects adults over the age of 55.It sometimes occurs in younger adults, but it almost never affectschildren. Acute myelogenous leukemia (also known as acute myeloidleukemia, or AML) occurs more commonly in adults than in children. Thistype of leukemia was previously called “acute nonlymphocytic leukemia.”Chronic myelogenous leukemia (CML) occurs mainly in adults. A very smallnumber of children also develop this disease.

Lymphoma is a type of cancer that originates in lymphocytes (a type ofwhite blood cell in the vertebrate immune system). There are many typesof lymphoma. According to the U.S. National Institutes of Health,lymphomas account for about five percent of all cases of cancer in theUnited States, and Hodgkin's lymphoma in particular accounts for lessthan one percent of all cases of cancer in the United States. Becausethe lymphatic system is part of the body's immune system, patients witha weakened immune system, such as from HIV infection or from certaindrugs or medication, also have a higher incidence of lymphoma.

In the 19th and 20th centuries the affliction was called Hodgkin'sDisease, as it was discovered by Thomas Hodgkin in 1832. Colloquially,lymphoma is broadly categorized as Hodgkin's lymphoma and non-Hodgkinlymphoma (all other types of lymphoma). Scientific classification of thetypes of lymphoma is more detailed. Although older classificationsreferred to histiocytic lymphomas, these are recognized in newerclassifications as of B, T, or NK cell lineage.

Autoimmune disease, or autoimmunity, is the failure of an organism torecognize its own constituent parts (down to the sub-molecular levels)as “self,” which results in an immune response against its own cells andtissues. Any disease that results from such an aberrant immune responseis termed an autoimmune disease. Prominent examples include Coeliacdisease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus(SLE), Sjögren's syndrome, multiple sclerosis (MS), Hashimoto'sthyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, andrheumatoid arthritis (RA).

Inflammatory diseases, including autoimmune diseases are also a class ofdiseases associated with B-cell disorders. Examples of autoimmunediseases include, but are not limited to, acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcalnephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, pamphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,perniciousanemia, rapidly progressive glomerulonephritis, psoriasis, andfibrosing alveolitis. The most common treatments are corticosteroids andcytotoxic drugs, which can be very toxic. These drugs also suppress theentire immune system, can result in serious infection, and have adverseaffects on the bone marrow, liver, and kidneys. Other therapeutics thathas been used to treat Class III autoimmune diseases to date have beendirected against T cells and macrophages. There is a need for moreeffective methods of treating autoimmune diseases, particularly ClassIII autoimmune diseases.

VIII. ARTIFICIAL ANTIGEN PRESENTING CELLS

In some cases, aAPCs are useful in preparing therapeutic compositionsand cell therapy products of the embodiments. For general guidanceregarding the preparation and use of antigen-presenting systems, see,e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S.Patent Application Publication Nos. 2009/0017000 and 2009/0004142; andInternational Publication No. WO2007/103009).

aAPCs are typically incubated with a peptide of an optimal length thatallows for direct binding of the peptide to the MHC molecule withoutadditional processing. Alternatively, the cells can express and antigenof interest (i.e., in the case of MHC-independent antigen recognition).In addition to peptide-MHC molecules or antigens of interest, the aAPCsystems may also comprise at least one exogenous assisting molecule. Anysuitable number and combination of assisting molecules may be employed.The assisting molecule may be selected from assisting molecules such asco-stimulatory molecules and adhesion molecules. Exemplaryco-stimulatory molecules include CD70 and B7.1 (B7.1 was previouslyknown as B7 and also known as CD80), which among other things, bind toCD28 and/or CTLA-4 molecules on the surface of T cells, therebyaffecting, for example, T-cell expansion, Th1 differentiation,short-term T-cell survival, and cytokine secretion such as interleukin(IL)-2 (see Kim et al., 2004, Nature, Vol. 22(4), pp. 403-410). Adhesionmolecules may include carbohydrate-binding glycoproteins such asselectins, transmembrane binding glycoproteins such as integrins,calcium-dependent proteins such as cadherins, and single-passtransmembrane immunoglobulin (Ig) superfamily proteins, such asintercellular adhesion molecules (ICAMs), that promote, for example,cell-to-cell or cell-to-matrix contact. Exemplary adhesion moleculesinclude LFA-3 and ICAMs, such as ICAM-1. Techniques, methods, andreagents useful for selection, cloning, preparation, and expression ofexemplary assisting molecules, including co-stimulatory molecules andadhesion molecules, are exemplified in, e.g., U.S. Pat. Nos. 6,225,042,6,355,479, and 6,362,001.

Cells selected to become aAPCs, preferably have deficiencies inintracellular antigen-processing, intracellular peptide trafficking,and/or intracellular MHC Class I or Class II molecule-peptide loading,or are poikilothermic (i.e., less sensitive to temperature challengethan mammalian cell lines), or possess both deficiencies andpoikilothermic properties. Preferably, cells selected to become aAPCsalso lack the ability to express at least one endogenous counterpart(e.g., endogenous MHC Class I or Class II molecule and/or endogenousassisting molecules as described above) to the exogenous MHC Class I orClass II molecule and assisting molecule components that are introducedinto the cells. Furthermore, aAPCs preferably retain the deficienciesand poikilothermic properties that were possessed by the cells prior totheir modification to generate the aAPCs. Exemplary aAPCs eitherconstitute or are derived from a transporter associated with antigenprocessing (TAP)-deficient cell line, such as an insect cell line. Anexemplary poikilothermic insect cells line is a Drosophila cell line,such as a Schneider 2 cell line (see, e.g. Schneider, J. Embryol. Exp.Morph. 1972 Vol 27, pp. 353-365). Illustrative methods for thepreparation, growth, and culture of Schneider 2 cells, are provided inU.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.

In one embodiment, aAPCs are also subjected to a freeze-thaw cycle. Inan exemplary freeze-thaw cycle, the aAPCs may be frozen by contacting asuitable receptacle containing the aAPCs with an appropriate amount ofliquid nitrogen, solid carbon dioxide (i.e., dry ice), or similarlow-temperature material, such that freezing occurs rapidly. The frozenaAPCs are then thawed, either by removal of the aAPCs from thelow-temperature material and exposure to ambient room temperatureconditions, or by a facilitated thawing process in which a lukewarmwater bath or warm hand is employed to facilitate a shorter thawingtime. Additionally, aAPCs may be frozen and stored for an extendedperiod of time prior to thawing. Frozen aAPCs may also be thawed andthen lyophilized before further use. Preferably, preservatives thatmight detrimentally impact the freeze-thaw procedures, such as dimethylsulfoxide (DMSO), polyethylene glycols (PEGs), and other preservatives,are absent from media containing aAPCs that undergo the freeze-thawcycle, or are essentially removed, such as by transfer of aAPCs to mediathat is essentially devoid of such preservatives.

In other preferred embodiments, xenogenic nucleic acid and nucleic acidendogenous to the aAPCs, may be inactivated by crosslinking, so thatessentially no cell growth, replication or expression of nucleic acidoccurs after the inactivation. In one embodiment, aAPCs are inactivatedat a point subsequent to the expression of exogenous MHC and assistingmolecules, presentation of such molecules on the surface of the aAPCs,and loading of presented MHC molecules with selected peptide orpeptides. Accordingly, such inactivated and selected peptide loadedaAPCs, while rendered essentially incapable of proliferating orreplicating, retain selected peptide presentation function. Preferably,the crosslinking also yields aAPCS that are essentially free ofcontaminating microorganisms, such as bacteria and viruses, withoutsubstantially decreasing the antigen-presenting cell function of theaAPCs. Thus crosslinking maintains the important APC functions of aAPCswhile helping to alleviate concerns about safety of a cell therapyproduct developed using the aAPCs. For methods related to crosslinkingand aAPCs, see for example, U.S. Patent Application Publication No.20090017000, which is incorporated herein by reference.

IX. KITS OF THE INVENTION

Any of the compositions described herein may be comprised in a kit. Insome embodiments, allogeneic CAR T-cells are provided in the kit, whichalso may include reagents suitable for expanding the cells, such asmedia, aAPCs, growth factors, antibodies (e.g., for sorting orcharacterizing CAR T-cells) and/or plasmids encoding CARs ortransposase.

In a non-limiting example, a chimeric receptor expression construct, oneor more reagents to generate a chimeric receptor expression construct,cells for transfection of the expression construct, and/or one or moreinstruments to obtain allogeneic cells for transfection of theexpression construct (such an instrument may be a syringe, pipette,forceps, and/or any such medically approved apparatus).

In some embodiments, an expression construct for eliminating endogenousTCR α/β expression, one or more reagents to generate the construct,and/or CAR⁺ T cells are provided in the kit. In some embodiments, thereincludes expression constructs that encode zinc finger nuclease(s).

In some aspects, the kit comprises reagents or apparatuses forelectroporation of cells.

The kits may comprise one or more suitably aliquoted compositions of thepresent invention or reagents to generate compositions of the invention.The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits may include at leastone vial, test tube, flask, bottle, syringe, or other container means,into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third, or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the chimeric receptor construct and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which thedesired vials are retained, for example.

X. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Clinical Application of Sleeping Beauty and Artificial AntigenPresenting Cells to Genetically Modify T Cells from Peripheral andUmbilical Cord Blood Materials and Methods

Isolation of Mononuclear Cells (MNC) from PB and UCB.

On or before Day 0, dilute PB with an equal volume and UCB with fourvolumes of PBS-EDTA. Slowly layer diluted blood (25 mL) onto Ficoll (12mL) in a 50 mL centrifuge tube(s) and centrifuge at 400×g for 30-40 min(no brake). Collect and transfer the mononuclear cell fraction(interface) using a transfer pipette to a fresh 50 mL centrifuge tube.Bring the volume up to 50 mL with PBS-EDTA and centrifuge at 450×g for10 min. Aspirate the supernatant and gently re-suspend the cellpellet(s) in 50 mL of Complete Culture Media (CCM). Centrifuge at 400×gfor 10 min Gently re-suspend and pool the cell pellets in CCM andperform a cell count using Trypan blue exclusion (Cellometer, PBMCprogram). MNC can be used for electroporation (Nucleofection) orcryopreserved for future use.

Preparation of T Cells for Electroporation on Day 0.

If using cryopreserved MNC, quickly thaw sufficient cells for a fullscale electroporation (2×10⁸ adding ˜20% to account for cell loss duringcentrifugation and 2 hr incubation) in a 37° C. water bath. Gentlyre-suspend and transfer cells to an appropriately sized centrifuge tubecontaining pre-warmed Complete Phenol-Free RPMI culture media (PF-RPMI),centrifuge at 200×g for 10 min (no brake), and aspirate the supernatant.Next, and if using freshly isolated MNC, re-suspend the MNC in PF-RPMI,perform a cell count (Cellometer) and transfer cells to an appropriatelysized cell culture vessel at a concentration of 10⁶ cells/ml. Incubatethe cells in a humidified 37° C./5% CO₂ incubator for 2 h±30 min.Transfer the MNC to a sterile centrifuge tube, spin at 200×g for 5 min(no brakes), aspirate the supernatant, and gently re-suspend and combinethe cell pellets in PF-RPMI. Perform a cell count (Cellometer) andcalculate the volume of the cell suspension required (2×10⁸ MNC).Transfer the calculated volume to a sterile 50 mL centrifuge tube andspin at 200×g for 10 min (no brake). Aspirate the supernatant so that noresidual media remains and gently re-suspend by tapping side of tube.

Electroporation (Nucleofection) of MNC (Full Scale Process Using 10Cuvettes) on Day 0.

Pre-incubate a sterile 12-well plate with 10 wells containing 4 mL ofwarm PF-RPMI in a humidified 37° C./5% CO₂ incubator. Prepare andpre-warm the Lonza Nucleofector Solution Human T cell kit (reconstitutedper manufacturer's instructions, www.lonza.com) to ambient temperaturein a Biosafety Cabinet (BSC). Prepare Nucleofector solution/DNA mastermix by adding 100 μL of supplemented Nucleofector solution, 15 μg oftransposon (supercoiled DNA plasmid designated as CD19RCD28/pSBSO), and5 μg of transposase (supercoiled DNA plasmid designated as pCMV-SB11)per reaction/cuvette. Disperse the MCN cell pellet by gently tapping theside of the centrifuge tube and re-suspend in Nucleofection solution/DNAmaster mix at a final cell concentration of 2×10⁷ cells/100 μL.Carefully transfer 100 μL of the cell suspension to each of ten LonzaNucleofection cuvettes, being careful to avoid bubbles. Tap the cuvetteonce, and electroporate using program U-014 (for unstimulated T cells).Transfer the cuvettes and the 12-well plate to the BSC. Harvest theelectroporated cells from each cuvette using an Amaxa fine tip transferpipette by adding ˜500 μL of the pre-warmed culture medium from thecorresponding well and return the plate to a humidified 37° C./5% CO₂incubator for 2 h±30 min. Following the 2-hr incubation, harvest andtransfer the cells from all wells to a sterile centrifuge tube. Washcells by centrifugation at 140×g for 8 min, ambient temperature, nobrake and aspirate and discard the supernatant so that no residualmedium covers the cell pellet. Disperse the cell pellet by gentlytapping the side of the centrifuge tube and gently re-suspend in CCM toachieve a single cell suspension. Perform cell count and adjust cellconcentration to 10⁶ cells/mL in CCM. Transfer the cell suspension tocell culture flask(s) and place in the incubator overnight. The sameprocess may be used for control EGFP-transfected cells (5×10⁶cells/cuvette with 5 μg Amaxa control EGFP supercoiled plasmid,pmaxGFP).

Analysis of CAR Expression by Flow Cytometry on Day 1 of the 1^(st) andSubsequence Stimulation Cycles.

Harvest the electroporated cells and perform a cell count using Trypanblue exclusion (Hemocytometer). Stain cells (1-2×10⁶) with antibodyspecific for CD3, CD4, CD8, and human IgG Fcγ as a measurement of CARexpression. Acquire cells on the FACS Calibur and analyze the data usingFCS Express software to calculate expression of CAR. Calculate CAR⁺cells in culture by the formula: (No. of Total viable cells)×(% CAR⁺cells)=No. of CAR⁺ cells.

Preparation of aAPC (Clone #4) on Day 1 of the 1^(st) and SubsequentStimulation Cycles.

aAPC (clone #4) was derived from K562 cells (parental line obtained fromAmerican Type Culture Collection) to co-express the desired T cellco-stimulatory molecule. Thaw an aliquot of frozen 100 Gy irradiatedaAPC in a 37° C. water bath. Cells are washed twice by centrifugation at400×g, 10 min in CCM and counted using Cellometer (Trypan blueexclusion). Calculate number of viable aAPC required for stimulation:(No. of CAR⁺ cells)×2=No. of irradiated aAPC required

aAPC-Mediated Stimulation of CAR⁺ T Cells on Day 1 Beginning of 1^(st)and Subsequent Stimulation Cycles.

Mix the electroporated cells (expressing CAR) and γ-irradiated aAPC(clone #4) in a sterile container at a ratio of 1:2 (CAR⁺ cell: viableaAPC) in CCM. Note that the aAPC ratio is adjusted for the expression ofCAR based on flow cytometry the day after electroporation. Add IL-21 (30ng/mL) to the cell suspension. Aliquot in T-75 cm² flask(s) and/orVueLife Culture bags at a concentration of 10⁶ cells/mL and return tothe incubator.

Continued Culture of CAR⁺ T Cells on Days 3 and 5.

Perform a half-media change, replenish IL-21, and maintain T cells at aconcentration of 10⁶ cells/mL.

End of First aAPC-Mediated Stimulation Cycle on Day 7.

Harvest cells, count, and stain for CD3, CD4, CD8, and Fcγ (CAR).

Depletion of CD56⁺ Cells Between 7 and 14 Days after Electroporation.

Perform a CD56 depletion using paramagnetic beads if CD56⁺CD3⁺lymphocytes ≧10%.

Recursive Addition of aAPC to Propagate T Cells to Clinically-SufficientNumbers During Stimulation Cycles #2, #3, & #4 Corresponding to Days8→14, Days 15→21, & Days 22→28.

Repeat the stimulation process up to 4 times. Add IL-2 (50 U/mL) to thecultures beginning on Days 7, 14 and 21, and then at each media change(three times a week, on a Monday-Wednesday-Friday schedule).Cryopreserve (archive) excess T cells as needed using a controlled ratefreezer for release testing and infusion.

Example 2 Clinical Application of Sleeping Beauty and Artificial AntigenPresenting Cells to Genetically Modify T Cells from Peripheral andUmbilical Cord Blood Results

Electro-transfer of DNA plasmids and propagation of T cells onγ-irradiated aAPC can be used to generate clinically-appealing numbersof T cells derived from PB and UCB for human applications. Thesegenetically modified T cells express an introduced CAR that recognizesthe TAA CD19, independent of major histocompatibility complex. TheSB-derived DNA plasmids to express the (i) transposon, a 2^(nd)generation CAR (CD19RCD28) that signals through CD28 and CD3-ε¹⁴, and(ii) transposase, SB11 (Jin et al., 2011), have been previouslydescribed (Singh et al., 2011; Davies et al., 2010; Kebriaei et al.,2012). The plasmids used in the current study were produced commerciallyby Waisman Clinical Biomanufacturing Facility (Madison, Wis.). The aAPC(clone #4), derived from K562 cells (parental line obtained fromAmerican Type Culture Collection), co-express desired T cellco-stimulatory molecules (each introduced molecule at 90% on cellsurface of aAPC), as previously described (Manuri et al., 2010). Here,CD19-specific T cells could be generated from mononuclear cells (MNC)derived from PB or UCB using SB transposition to introduce the CARfollowed by addition of aAPC to numerically expand the T cells in aCAR-dependent manner (FIGS. 1 and 4) (Singh et al., 2011; Singh et al.,2008). Ten cuvettes (2×10⁷ MNC/cuvette) were electroporated for eachrecipient using 15 μg of DNA plasmid (CD19RCD28/pSBSO) coding fortransposon (CAR) and 5 μg of DNA plasmid (pCMV-SB11) coding fortransposase (SB11). The number of cuvettes can be reduced if MNC arelimiting or scaled back for laboratory work. The day of electroporationis defined as “Day 0” of Stimulation cycle #1. As controls for flowcytometry and culture conditions, autologous T cells are mockelectroporated (without DNA plasmid) and numerically expanded onγ-irradiated aAPC (clone #4) that had been pre-loaded with OKT3 tocross-link CD3 to sustain T cell proliferation. The efficiency ofelectrotransfer and viability of the T cells the day afterelectroporation was routinely assessed (FIG. 2B). The expression of EGFPfrom control DNA plasmid (designated pmaxGFP) and CAR at this initialtime point reflects protein expression from the integrated and episomalplasmid. Typically, EGFP expression was measured at 60% the day afterelectroporation and CAR expression at ˜40% (FIG. 2A) with T cellviability between 40%-50%. Recursive additions of γ-irradiated aAPC inthe presence of soluble recombinant human IL-2 and IL-21 retrieve Tcells stably expressing CAR (CD19RCD28). CD3^(neg)CD56⁺ NK cells aredepleted from the culture using CD56-specific paramagnetic beads if thepercentage of these NK cells is ≧10% and especially if the percentage ofCAR expressed on the T cells is low. This depletion prevents the rapidovergrowth of NK cells which interferes with the ability of aAPC tosustain the proliferation of CAR⁺ T cells. On occasion, depletion of NKcells from CAR⁺ T cells is undertaken during the last two stimulationcycles, but this introduces a loss of desired cells due to co-expressionof CD56 on some CAR⁺ T cells. The T cells were grown in a functionallyclosed system using VueLife culture bags past Day 14. A subset of thegenetically modified and propagated T cells are typically cryopreservedat Day 14 or Day 21 (end of Stimulation cycles #2 or #3) of co-cultureon aAPC to serve as a source of archived material for future analysesand to be thawed if unanticipated problems subsequently occur during themanufacturing process. T cells are typically harvested on or about Day28 of culture (FIG. 3) that routinely express >90% CAR and are >80%viable (FIGS. 2C, D). After four weeks of co-culture on aAPC, theaverage fold-expansion of CD3⁺ T cells is 19,800±11,313 with CAR⁺expression being 90%±7.5% (Singh et al., 2011). These T cells arecryopreserved and undergo in-process and release testing that informs onthe safety and therapeutic potential of the manufactured product.Release testing is undertaken in compliance with clinical laboratoryimprovement amendments (CLIA) to generate a certificate of analysisprior to infusion into recipients on clinical trials.

Example 3 Manufacture of Clinical-Grade CD19-Specific T Cells StablyExpressing Chimeric Antigen Receptor Using Sleeping Beauty andArtificial Antigen Presenting Cells Materials and Methods

Generation of Clinical-Grade DNA Plasmids.

The SB transposon, CoOpCD19RCD28/pSBSO, expresses the human codonoptimized (CoOp) 2^(nd) generation _(CoOp)CD19RCD28 CAR (SEQ ID NO: 1)under EF-1/HTLV hybrid composite promoter (InvivoGen) comprised ofElongation Factor-1α (EF-1α) (Kim et al., 1990) and 5′ untranslatedregion of the Human T-Cell Leukemia Virus (HTLV) (Singh et al., 2011;Davies et al., 2010). The derivation of this DNA plasmid is described inFIG. 10. The SB transposase, SB11, under the cytomegalovirus (CMV)promoter was expressed in cis from the DNA plasmid pCMV-SB11 (Singh etal., 2011). The derivation of this DNA plasmid is described in FIG. 11.Both plasmids were sequenced in their entirety and manufactured byWaisman Clinical Biomanufacturing Facility (Madison, Wis.) usingkanamycin for selection of the bacterial strain E. coli DH5α. Therelease criteria for the DNA plasmids are shown in Table 1. CD19 wasexpressed using the DNA plasmid ΔCD19_(CoOp)-F2A-Neo/pSBSO (FIG. 12).

TABLE 1 Release criteria for DNA plasmids coding for SB transposon andtransposase. Test Specification Appearance Clear colorless liquidRestriction Mapping & Digest with Nde I; AflIII & NheI; HindIII AgaroseGel & AvrII; Xba & NcoI for transposon. Digest with NdeI; XhoI & PvuII;AflIII; NcoI for transposase Sequencing Sequence conforms to originalcoding Concentration via absorbance 2.0 ± 0.2 mg/mL A260/A280 absorbanceratio 1.8-2.0 Kinetic LAL test for Bacterial <50 EU/mg Endotoxin PlasmidForm (% supercoiled >90% supercoiled via HPLC) Sterility Test No growthobserved E. coli host protein via <0.3% ELISA E. coli RNA via HPLC  <10%

Cell Counting.

Trypan-blue exclusion was used to distinguish live from dead cells andcounted using Cellometer (Nexecelom Bioscience) (Singh et al., 2011).

Isolation of PBMC.

Leukapheresis products from two male volunteer healthy donors werepurchased from Key Biologics LLC (Memphis, Tenn.). The peripheral bloodmononuclear cells (PBMC) were isolated by adapting the Biosafe Sepaxsystem (Eysins, Switzerland) for work in compliance with cGMP. Briefly,after closing all the clamps on the CS-900 kit, 100 mL Ficoll (GEHealthcare) was aseptically transferred via 60 mL syringes to a densitygradient media bag (“ficoll bag”) via Luer-lock connector and the tubingwas heat sealed using a hand held sealer (Sebra, Model#2380). The kitwas spike-connected to a 1,000 mL bag containing CliniMACS buffer(PBS/EDTA, Miltenyi, Cat#70026) with 20 mL 25% Human Serum Albumin (HSA)(Baxter) (2% v/v, wash buffer) for washes, a final product bag [300 mLTransfer Pack with Coupler (Baxter/Fenwal 4R2014)] and a reagent/bloodbag. Using the density gradient-based separation protocol (v126), thesyringe piston was loaded into the centrifuge chamber and the cover ofthe Sepax unit closed. The reagent/blood bag and product bags were hungand all stopcocks were seated on the rotary pins in the ‘T’ position.After connecting the pressure-sensor line, the kit was validated byautomatic single-use test and then manually primed using gravity flow.After completion of the cycle, the final product was asepticallytransferred into a centrifuge tube and washed once each with wash bufferand phosphate buffered saline (PBS) at 400 g for 10 minutes. Aftercounting, cells were cryopreserved using cryopreservation media (50%HSA, 40% Plasmalyte, 10% DMSO) in CryoMACS Freeze bags (Miltenyi) andvials (Nunc) using BM5 program (4° C. to −4° C. at rate −2° C./min, −4°C. to −60° C. at rate −35° C./min, −60° C. to −20° C. at rate 8° C./min,−20° C. to −45° C. at rate −2.5° C./min, −45° C. to −80° C. at rate −10°C./min) in a controlled-rate freezer (Planer Kryo 750).

Manufacture of aAPC (Clone #4) Master and Working Cell Banks.

K562 were transduced by lentivirus at the University of Pennsylvania togenerate aAPC (clone #4, designated CJK64.86.41BBL.GFP.IL-15.CD19) thatco-express (i) CD19, (ii) CD64, (iii) CD86, (iv) CD137L, and (v)membrane bound IL-15 (mIL15) as a bi-cistronic vector with EGFP. TheaAPC were numerically expanded in HyQ RPMI 1640 (Hyclone) containing 10%heat-inactivated defined FBS (Hyclone) and 2 mM Glutamax-1 (LifeTechnologies-Invitrogen) culture media (CM) maintaining the cells at5×10⁵ cells/mL. A master cell bank (MCB) of 320 vials was producedthrough Production Assistance of Cellular Therapies (PACT) (Table 2). A200 vial working cell bank (WCB) of Clone 4 aAPC derived from the MCBwas then generated at MDACC and tested (Table 3).

TABLE 2 Release criteria for K562-derived aAPC (clone #4) master cellbank. Test Specification Replication-competent Lentivirus NegativeProduct Enhanced Reverse Transcriptase for Negative Detection ofRetrovirus Endotoxin LAL Negative Agar Cultivable and Non-AgarCultivable Negative Mycoplasma HIV-1/2 Proviral DNA by PCR Negative HBVDNA by PCR Negative HCV RNA by RT-PCR Negative CMV DNA by PCR NegativeParvovirus B19 Negative HTLV-I/II Proviral DNA by PCR Negative EBV DNAby PCR Negative HHV-6 DNA by PCR Negative HHV-7 DNA by PCR NegativeHHV-8 DNA by PCR Negative Adeno-associated Virus Negative In vivoInapparent Virus Negative Negative Bovine Virus by 9CFR Negative PorcineVirus by modified 9CFR PT-1 Negative Adeno-associated Virus NegativeIsoenzyme analysis Human Origin Morphology by Transmission Electron Noidentifiable Microscopy virus-like particles nor any other microbialagents Immunophenotyping: CD19 90% CD64 90% CD86 90% CD137L 90%EGFP-mIL15 90% Bacteriostasis & Fungistasis Negative Sterility by21CFR610.2 Negative

TABLE 3 Release criteria for K562-derived aAPC (clone #4) working cellbank. TEST LABORATORY SPECIFICATION Bacteriostasis and FungistasisAppTec Laboratory Negative Services Sterility by 21CFR610.12 AppTecLaboratory Negative Services Agar Cultivable and BioReliance NegativeNon-Agar Cultivable Mycoplasma In vitro Adventitious Virus BioRelianceNegative testing Identity Isoenzyme analysis BioReliance Human OriginPhenotype MDACC GMP Flow ≧80% GFP⁺ Cytometry Laboratory ≧80% CD19⁺ ≧80%CD86⁺ ≧80% CD137L⁺ ≧80% CD64⁺ ≧80% CD32⁺ Endotoxin (LAL) EndoSafe MDACCGMP Quality ≦5 EU/mL Control Laboratory Viability by Trypan Blue MDACCGMP Quality ≧60% Control Laboratory

aAPC (Clone #4) to Selectively Propagate CAR⁺ T Cells.

The γ-irradiated aAPC were used to numerically expand the geneticallymodified T cells. Thawed aAPC from WCB were propagated in CM for up to60 days in VueLife cell culture bags and harvested using Biosafe SepaxII harvest procedure. Briefly, a CS-490.1 kit was connected to a 300 mLoutput bag (transfer pack) via Luer lock connection. The separationchamber was installed in the pit and the tubing was inserted into theoptical sensor and stopcocks aligned in the T position. After connectingthe pressure sensor line, the product bag and supernatant/plasma bagswere hung on the holder. The modified protocol PBSCv302 was selectedfrom the Sepax menu and the volume of input product to be processed(initial volume) was set to ≦840 mL. After validation and kit test, theprocedure was started. Following completion, the bags were removed,clamps closed and the kit was removed. The cells from the final productbag were aseptically removed, washed twice with wash media (10% HSA inPlasmalyte) and counted. aAPC were irradiated (100 Gy) using a CIS BIOInternational radiator (IBL-437 C#09433) and cryopreserved for later usein cryopreservation media using a controlled-rate freezer (Planer Kryo750).

OKT3-Loading of aAPC.

The OKT3-loaded (via CD64) aAPC (clone#4) were used to propagate control(CAR^(neg)) autologous control T cells that had not undergone geneticmodification. The aAPC, obtained from culture, were incubated overnightin serum-free X-Vivo 15 (cat #04-744Q, Lonza) containing 0.2% acetylcysteine (Acetadote, Cumberland Pharmaceuticals) termed Loading Medium(LM). The next day cells were washed, irradiated (100 Gy) using a GammaCell 1000 Elite Cs-137 radiator (MDS Nordion), resuspended in LM at aconcentration of 10⁶ cells/mL along with 1 μg/10⁶ cells of functionalgrade purified anti-human CD3 (clone-OKT3, 16-0037-85, eBioscience) andincubated with gentle agitation on a 3-D rotator (Lab-Line) at 4° C. for30 minutes. Following three washes with LM the cells were used inexperiments or frozen in aliquots in liquid nitrogen in the vapor layerfor later use.

Manufacture of CAR^(P) T Cells.

Thawed PBMC were resuspended in (i) Human T-cell kit (cat# VPA-1002,Lonza; 100 μL for 2×10⁷ cells in one cuvette), with (ii) the DNA plasmid(_(CoOp)CD19RCD28/pSBSO) coding for CD19RCD28 CAR transposon (15 μgsupercoiled DNA per 2×10⁷ PBMC per cuvette), and (iii) the DNA plasmid(pCMV-SB11) coding for SB11 transposase (5 μg supercoiled DNA per 2×10⁷PBMC per cuvette). This mixture was immediately transferred to a cuvette(Lonza), electroporated (defining culture day 0) using Nucleofector II(Amaxa/Lonza), rested in 10% RPMI complete media for 2 to 3 hours, andafter a half-media change, incubated overnight at 37° C., 5% CO₂. Thefollowing day, cells were harvested, counted, phenotyped by flowcytometry, and co-cultured with γ-irradiated aAPC at a ratio of 1:2(CAR⁺ T cell:aAPC), which marked culture day 1 and the beginning of a7-day stimulation cycle. IL-21 (cat # AF-200-21, PeproTech) and IL-2(cat # NDC 65483-116-07, Novartis) were added on aMonday-Wednesday-Friday schedule onwards of day 1 and day 7,respectively. NK cells can prevent the numeric expansion of CAR⁺ Tcells, especially if their overgrowth occurs early in the tissueculturing process. Therefore, CD56-depletion was performed ifCD3^(neg)CD56⁺ cells ≧10% using CD56 beads (cat #70206, MiltenyiBiotech, 20 μL beads/10⁷ cells) on LS columns (cat #130-042-401,Miltenyi Biotech) in CliniMACS buffer containing 25% HSA (80 μL/10⁷cells). T cells were cryopreserved as backup on culture day 21 afterelectroporation and the end of the 3^(rd) stimulation cycle using acontrolled-rate freezer (Planer Kryo 750) as described above, and storedin liquid nitrogen (vapor-layer). The cell counts for total, CD3+, andCAR⁺ T cells were plotted over time and slopes determined using linearregression. The fold-expansion results were compared using a Student'st-test. CD4/CD8 ratios were calculated for each time point andvalidation runs and averaged.

Generation of CAR^(neg) Control T Cells.

As a control, 5×10⁶ mock transfected PBMC were co-cultured withirradiated and anti-CD3 (OKT3) loaded K562-derived aAPC clone #4 at aratio of 1:1 in a 7-day stimulation cycle. All the cultures weresupplemented with IL-21 (30 ng/mL) from culture day 1 onwards, and IL-2(50 U/mL) starting 7 days after the start of the culture. All cytokineswere subsequently added on a Monday-Wednesday-Friday schedule.

Cell Lines.

CD19⁺ Daudiβ₂m [Burkitt lymphoma, co-expressing β2 microglobulin,(Rabinovich et al., 2008)] and CD19⁺ NALM-6 (pre-B cell) were culturedas described previously (Singh et al., 2011). EL-4 cells (mouse T-celllymphoma line) from ATCC were modified to express CD19 using theconstruct ΔCD19_(CoOp)-F2A-Neo/pSBSO. Briefly, 5×10⁶ EL-4 cells wereresuspended in 100 μL, of Amaxa Mouse T cell Nucleofector kit (Catalogue#VPA-1006) with SB transposon (ΔCD19_(CoOp)-F2A-Neo/pSBSO, 3 μg) and SBtransposase (pCMV-SB11, 1 μg) and electroporated (program X-001) usingNucleofector II (Lonza). The transfectants were cultured in a cytocidalconcentration of G418 (0.8 mg/mL) and underwent fluorescent activatedcell sorting (FACS) for homogeneous expression of CD19 to obtain a clone(clone #17). Jurkat cell were obtained from ATCC and electroporated(Program T-14, Nucleofector II, Lonza) with _(CoOp)CD19RCD28mz(CoOp)/pSBSO using the Amaxa/Lonza Nucleofector solution (Kit V). Twoweeks after electroporation, Jurkat cells stably expressing CARunderwent FACS for homogeneous expression of CAR to obtain a clone(clone #12) (Maiti et al., 2013). Cell lines were maintained in HyQ RPMI1640 (Hyclone) supplemented with 2 mM Glutamax-1 (Invitrogen) and 10%heat-inactivated Fetal Calf Serum (FCS) (Hyclone; 10% RPMI). All celllines were validated using STR profiling or karyotyping according toinstitutional cell line authentication policy.

Immunophenotype of Cells.

Cells were stained using antibodies (Table 4) in 100 μL FACS Buffer (2%FBS, 0.1% Sodium Azide) for 30 minutes at 4° C. For intracellularstaining, after fixing/permeabilization for 20 minutes, cells werestained in perm wash buffer with appropriate antibodies for 30 minutesat 4° C. Acquisition was performed using FACSCalibur (BD Bioscience) andanalyzed using Cell Quest BD Bioscience) or FCS Express 3.00.0612 (DeNovo Software, Thornhill, Ontario, Canada).

TABLE 4 Antibodies used for flow cytometry. Catalogue CellsAntibody/Fluorochrome Vendor No. T cells CD3-PE BD Biosciences 347347CD4-APC BD Biosciences 340443 CD8-PerCPCy5.5 BD Biosciences 341051CD16-PE BD Biosciences 347616 CD25-APC BD Biosciences 555434CD28-PerCPCy5.5 BD Biosciences 337181 CD32-FITC BD Biosciences 555448CD39-FITC eBioscience 11-0399-42 CD45TA-APC BD Biosciences 550855CD45RO-PE BD Biosciences 555489 CD57-FITC BD Biosciences 555619 CD56-APCBD Biosciences 555518 CD62L-PE BD Biosciences 555544 CD69-PE BDBiosciences 555531 CD127 (IL-7Ra) - Alexa Fluor BD Biosciences 558598647 CD150-PE BD Biosciences 559592 CD279 (PD-1) -PE BD Biosciences557946 CCR7-PerCPCy5.5 BioLegend 335605 Granzyme B-Alexa Fluor 647 BDBiosciences 560212 HLA-DR-PerCPCy5.5 BD Biosciences 339205 Perforin-FITCBD Biosciences 556577 Anti-human Fcγ-PE Invitrogen H10104 K562 aAPC(Clone CD19-PE BD Biosciences 555413 #4) CD19-APC BD Biosciences 555415CD64-PE BD Biosciences 558592 CD86-PE BD Biosciences 555658 CD137L-PE BDBiosciences 559446 F(ab′)2 fragment of Goat anti- Jackson 115-116-072Mouse IgG, F(ab′)2 fragment Immunoresearch specific-PE (For OKT3loading)

Western Blot.

Protein expression of the chimeric CD3-ζ (73-kDa) derived from CD19RCD28was assessed as described previously (Singh et al., 2008). Briefly,protein lysates were transferred using iBlot Dry Blotting System(Invitrogen) onto nitrocellulose membrane, incubated with mouseantihuman CD3-ζ monoclonal antibody (cat #551033, 0.5 μg/mL, BDBiosciences, CA) followed by horseradish peroxidase (HRP)-conjugatedgoat anti-mouse IgG (cat #1858413, 1:10,000; Pierce, Ill.), developedusing SuperSignal West Femto Maximum Sensitivity substrate (Pierce,Ill.) and chemiluminescence captured using VersaDoc™ 4000 geldocumentation system (BioRad, CA).

Telomere Length Analysis by Fluorescence In Situ Hybridization and FlowCytometry (Flow-FISH).

Telomere length of the T cells was measured by using the Telomere PNAKit/FITC for Flow Cytometry (DAKO) according to the manufacturer'sinstructions. Briefly, isolated cells (CD4 or CD8) and control cells(cat #85112105, CEM-1301 cell line; ECACC) were mixed in equal measurein hybridization solution with or without FITC-labeled telomere PNAprobe for 10 minutes at 82° C.; hybridized overnight in the dark at roomtemperature; washed twice with a wash solution at 40° C.; resuspended inPBS containing 2% FCS and propidium iodide (1 μg/mL); and analyzed on aFACSCalibur (BD Biosciences). FITC-labeled fluorescent calibration beads(cat #824A, Quantum™ FITC MESF, Bangs Laboratories) were used forcalibration of the flow cytometry machine. Relative telomere length(RTL) was determined by comparing T cells with a CEM-1301 cell line per:

${RTL} = \frac{\; {\; \begin{matrix}\left( {{{Mean}\mspace{14mu} {FL}\; 1\mspace{14mu} {sample}\mspace{14mu} {cells}\mspace{14mu} {with}\mspace{14mu} {probe}} -} \right. \\{\left. {{Mean}\mspace{14mu} {FL}\; 1\mspace{14mu} {sample}\mspace{14mu} {cells}\mspace{14mu} {without}\mspace{14mu} {probe}} \right) \times 2 \times 100}\end{matrix}}\;}{\begin{pmatrix}{{{Mean}\mspace{14mu} {FL}\; 1\mspace{14mu} {reference}\mspace{20mu} {cells}\mspace{14mu} {with}\mspace{14mu} {probe}} -} \\{{Mean}\mspace{14mu} {FL}\; 1\mspace{14mu} {reference}\mspace{14mu} {cells}\mspace{14mu} {without}\mspace{14mu} {probe}}\end{pmatrix}}$

Chromium Release Assay.

T cells were evaluated for their cytotoxicity in a standard 4-hourchromium release assay using ⁵¹Cr-labeled target cells. T cells wereplated in triplicate at 1×10⁵, 0.5×10⁵, 0.25×10⁵, 0.125×10⁵ and0.0625×10⁵ cells/well with 5×10³ target cells in a 96-well V-bottomplate (Costar). After incubation, 50 μL, of supernatant was harvestedonto a LumaPlate (Perkin Elmer), read in TopCount NXT (Perkin Elmer),and percent specific lysis was calculated per:

$\frac{{{Experimental}\mspace{14mu} 51{Cr}\mspace{14mu} {released}} - {{Spontaneous}\mspace{14mu} 51{Cr}\mspace{14mu} {released}}}{{{Maximum}\mspace{14mu} 51{Cr}{\mspace{11mu} \;}{released}} - {{Spontaneous}\mspace{14mu} 51{Cr}\mspace{14mu} {released}}} \times 100$

Spontaneous and maximum release was determined by measuring chromium inthe conditioned supernatant from target cells incubated with CM or 0.1%Triton X-100 (Sigma), respectively.

Endotoxin Testing.

Endotoxin level in final products was determined using Endosafe®-PTSPortable Test System (Charles River Laboratories) as per themanufacturer's guidelines. The test has a detection limit of 0.01-10EU/mL, which can be converted to EU/patient weight.

Mycoplasma Testing.

Mycoplasma detection by PCR was performed using the TaKaRa MycoplasmaDetection Kit (Clontech) according to manufacturer's instructions.

T-Cell Receptor Vβ Repertoire.

T-cell receptor (TCR)-Vβ usage of culture day 28 and day 35 CAR⁺ T cellswas determined using a panel of 24 TCR Vβ-specific mAbs (cat # IM3497,IO TEST Beta Mark TCR-Vβ repertoire kit, Beckman Coulter) used inassociation with CD3-specific mAb (cat #340949, BD Biosciences, 10 μL)and isotype-matched control mAbs (cat #552834, BD Biosciences).

Real-Time PCR to Determine Copy Number of Integrated CAR.

To determine the copy number of integrated CD19RCD28 CAR in geneticallymodified T cells, 50-100 ng genomic DNA (cat #80204, AllPrep DNA/RNAMini Kit, Qiagen) was amplified using Steponeplus real-time PCR system(Applied Biosystems) in a PCR reaction (2 min at 50° C., 10 min at 95°C., followed by 40 cycles of 15 sec at 95° C. and 1 min at 60° C.) usingthe following primers: forward (5′-CAGCGACGGCAGCTTCTT-3′; SEQ ID NO: 8),reverse (5′-TGCATCACGGAGCTAAA-3′; SEQ ID NO: 9) and probe(5′-AGAGCCGGTGGCAGG-3′; SEQ ID NO: 10). Primers (Cat #4316844, AppliedBiosystems) for RNAse P gene was used as an internal control.Serially-diluted genomic DNA from a genetically modified Jurkat-cell(clone #12) containing 1 copy of CAR from CD19RCD28mz (CoOp)/pSBSO DNAplasmid was used to generate a standard curve (Maiti et al., 2013). Allthe primers, probes and TaqMan Gene Expression Master Mix were purchasedfrom Applied Biosystems.

PCR for SB11 Transposase.

DNA (20 ng) (AllPrep DNA/RNA Mini Kit, Qiagen) isolated from CAR⁺ Tcells was amplified using a thermal cycler (PTC-200, DNA Engine, BioRad)using forward (5′-ATGGGAAAATCAAAAGAAATC-3′; SEQ ID NO: 11) and reverse(5′-CTAGTATTTGGTAGCATTGC-3′; SEQ ID NO: 12) primers in a PCR reaction(95° C. for 5 min; 25 cycles of 95° C. for 15 sec, 58° C. for 40 sec,72° C. for 60 sec; followed by a final extension at 72° C. for 7 min)GAPDH was used as the housekeeping gene and was amplified in the samePCR reaction using the primers, forward (5′-TCTCCAGAACATCATCCCTGCCAC-3′;SEQ ID NO: 13) and reverse (5′-TGGGCCATGAGGTCCACCACCCTG-3′; SEQ ID NO:14). Linearized pCMV-SB11 plasmid DNA (1 ng) and genomic DNA (20 ng)from genetically modified Jurkat cells stably expressing SB11 and EGFP(expressed from DNA plasmid SB11-IRES2-EGFP) (Maiti et al., 2013) wereused as a positive control. Mock electroporated (no DNA) andOKT3-aAPC-propagated T cells were used as a negative control.

Assay to Assess for Unwanted Autonomous T-Cell Growth.

To monitor aberrant T-cell growth, 2×10⁵ CAR⁺ T cells, harvested after 4aAPC-mediated stimulation cycles (28 days after electroporation) werecultured in triplicate in a 24-well tissue culture plate for anadditional 18 days. (i) Positive control: the presence of aAPC andcytokines (50 U/mL IL-2 and 30 ng/mL IL-21). (ii) Test: the absence ofaAPC and cytokines. The genetically modified T cells passed the assaywhen total viable cells at day 18 were (i) >2×10⁵ cells for CAR⁺ T cellscultured with aAPC and cytokines and (ii) <2×10⁴ cells for CAR⁺ T cellscultured without aAPC and cytokines.

G-Band Karyotyping.

CAR⁺ T cells at the end of co-culture were harvested and the slides werestained using Giemsa stain using standard procedure. A total of 20G-banded metaphases were analyzed.

Example 4 Manufacture of Clinical-Grade CD19-Specific T Cells StablyExpressing Chimeric Antigen Receptor Using Sleeping Beauty andArtificial Antigen Presenting Cells Results

aAPC (Clone #4).

K562 functioning as aAPC (clone #4) were employed to selectivelypropagate CAR⁺ T cells. The cultured aAPC were harvested from VueLifebags by a Sepax II device using the volume reduction procedure, whichtook 40 minutes. The mean preprocessing volume and aAPC counts were 575mL (range 500-700 mL) and 4.9×10⁸ (range 3.2×10⁸ to 7.7×10⁸),respectively. After processing with Sepax II, mean recovery of cells was108% (range 75% to 136%), with an output volume of 125 mL (range 50-200mL) resulting in mean volume reduction of 78.3% (range 60% to 91.6%,FIGS. 5A, B). The automated cell recovery was similar to a manual volumereduction procedure (82%), which took 45 minutes of sustained operatortime and resulted in 91% volume reduction. aAPC were regularly monitoredby flow cytometry for >80% expression of the introduced transgenescoding for CD19, CD64, CD86, CD137L, and EGFP (as a marker forexpression of mIL15). The immunophenotyping was undertaken upongenerating the MCB and WCB and upon each addition of γ-irradiated aAPCto T-cell cultures (that marked the beginning of each stimulation cycle,FIG. 5C). MCB and WCB for Clone 4 aAPC tested negative for sterility andmycoplasma on cells and cell supernatant. In the biosafety testing, novirus was detected by adventitious virus testing, replication competentretrovirus testing, and screening for a range of human pathogenicviruses. Testing validated that the aAPC (clone #4) was derived fromK562 based on finger printing (Table 5).

TABLE 5 STR fingerprinting of K562 aAPC (Clone #4). STR K562 aAPC (Clone4) AMEL X CSF1PO  9, 10 D13S317  8 D16S539 11, 12 D18S51 15, 16 D19S43314, 14.2 D21S11 29, 30, 31 D2S1338 17 D3S1358 16 D5S818 11 D7S820  9, 11D8S1179 12 FGA 21 TH01   9.3 TPOX 8, 9 vWA 16

Manufacture of CAR⁺ T Cells.

Validation studies were undertaken for large scale production ofCD19-specific CAR⁺ T cells to establish that PBMC can be electroporatedand propagated to clinically meaningful numbers (Huls et al., 2013)(FIG. 6) and meet pre-established release criteria (Table 6). Two normaldonor apheresis products were processed to isolate mononuclear cells(MNC) using the Sepax cell-processing system. The apheresis products 201mL (donor 1) and 202 mL (donor 2) were processed in two batches (˜100mL/batch) generating a 50 mL output product. The preprocessing countswere similar to post-processing counts of the apheresis products. Atotal of 5.3×10⁹ and 7.1×10⁹ cells were isolated containing 40.5% and51% CD3⁺ T cells respectively from donor 1 and donor 2. Cells were thencryopreserved in aliquots (4×10⁷ cells/mL) in CryoMACS freeze bags (10mL) and reference cryovials (1 mL) for later use. Three separatevalidation experiments were performed and are summarized in Table 6. Forvalidation run 1 and 2 (V1, V2) cells from donor 1 and for validation 3(V3) cells from donor 2 were used. For each run freshly-thawed PBMC wereelectroporated and ex vivo numerically expanded in separate culturingprocedures (Table 7). On culture day 0, 3×10⁸ (V1) and 8×10⁸ (V2, V3)cells were thawed (viability, 88.9% to 97.6%) and rested for 2 hoursprior to electroporation. 2 to 3×10⁸ cells (V1=2×10⁸; V2 and V3=3×10⁸)were electroporated at 2×10⁷ cells per cuvette withCD19RCD28mz(CoOp)/pSBSO transposon and pCMV-SB11 transposase DNAplasmids and the following day (culture day 1) co-cultured with aAPCclone #4 based on CAR expression. The electroporation efficiency for thethree validation runs was assessed on culture day 1 as measured byexpression of CAR (33.7%, 25.5% and 47.1%). CAR expression at the end ofco-culture (culture day 28) was 92%, 99.2%, and 96.7%, and the culturescontained mean 95%±5.3% CD3⁺ T cells with negligible amounts ofcontaminating CD19⁺ cells (mean=0.7%±0.15%) and CD32⁺ aAPC(mean=0.6%±0.6%, FIG. 7A, Table 6). The inventors further confirmed CARexpression by Western blot of whole-cell lysates ofelectroporated/propagated T cells using CD3-ζ chain-specific mAbrevealing an expected 73-kDa chimeric ζ chain (Singh et al., 2008) (FIG.7B). Upon inspection of the kinetics of T-cell growth on aAPC, theinventors observed an accelerated rate of T-cell propagation at the endof the second week of culture (end of stimulation cycle 2), which isconsistent with increased fold-expansion of total (p=0.01) and CD3⁺(p=0.01) T cells as compared to the fold-expansion in the first week ofstimulation. The weekly fold-expansion at the end of the third andfourth week for total (p=0.01, p<0.001), CD3⁺ (p=0.03, p<0.001), CAR⁺(p=0.02, p<0.001) T cells was consistently higher than that of week-one,respectively (FIG. 13).

TABLE 6 Acceptance criteria for releasing electroporated and propagatedT cells Results Validation Run Validation Run Validation Run TestSpecification #1 #2 #3 Sterility - Negative (No No growth at 14 Nogrowth at 14 No growth at 14 Bacteria and growth at 14 days days daysFungi days) Mycoplasma Negative by PCR Negative Negative Negative VisualNo evidence of No evidence of No evidence of No evidence of Inspectioncontamination contamination contamination contamination Viability ≧70%99% 99% 98% (Trypan Blue or 7AAD) Gated ≧80% CD3⁺ 89% CD3⁺ 97% CD3⁺ 99%CD3⁺ Immunophenotyping ≧10% CAR⁺ 92% CAR⁺ 99% CAR⁺ 96% CAR⁺ <5% CD32⁺1.3% CD32⁺ 0.04% CD32⁺ 0.51% CD32⁺ <5% CD19⁺ 0.75% CD19⁺ 0.49% CD19⁺0.78% CD19⁺ Endotoxin LAL Endotoxin lebel <0.004 EU/mL <0.667 EU/mL<0.089 EU/mL <5 EU/recipient weight (for validation, per mL) Screen for<2 × 10⁴ cells/mL <2 × 10⁴ cells/mL <2 × 10⁴ cells/mL <2 × 10⁴ cells/mLUnwanted for the cells Autonomous without Growth cytokines or aAPC atDay 18

TABLE 7 Characterization of T cells before and after co-culture onγ-irradiated aAPC. Aphersis Day 1 after electroporation Day 28 ofco-culture CD4: CD3⁺ CD3⁺ CD4: CD3⁺ CD4⁺ CD8⁺ CD4: CD3⁺ CD4⁺ CD8⁺ Via-Fold Expt. CD3 CD8 CD4⁺ CD8⁺ CD3 CD8 CAR CAR⁺ CAR⁺ CAR⁺ CD3 CD8 CAR CAR⁺CAR⁺ CAR⁺ bility exp* V1 40.5 1.65 18.5 11.2 64.7 2.4 33.7 41.0 28.011.1 88.9 0.02 92.0 97.7 2.46 87.2 99% 82.6 V2 59.8 4.1 25.5 17.1 25.93.8 97.1 0.01 99.2 97.4 1.77 91.2 99% 536.7 V3 51.2 1.59 29.1 18.3 87.72.3 47.1 45.0 20.1 31.5 99.2 0.8 96.0 96.0 43.4 51.4 98% 561.2 *Total(inferred) viable cells

The inventors observed similar weekly fold-expansion for CD3⁺ and CAR⁺ Tcells past week one of stimulation. After 4 weeks of co-culture onγ-irradiated aAPC there was an average 545-fold numeric expansion ofCD3⁺ T cells with a 1,111-fold expansion of CAR⁺ T cells. The ex vivoexpansion (culture day 28) resulted in an average 2.86×10¹⁰ CD3⁺ Tcells, almost all of which were CAR⁺ (2.65×10¹⁰). The propagationkinetics of total (p=0.18), CD3⁺ (p=0.17) and CAR⁺ (p=0.2) T cells forthe three validation runs were similar (FIGS. 8A, B, C). These datasupport the recursive addition of aAPC for the selective outgrowth ofCD19-specific T cells.

Immunophenotype of Electroporated and Propagated CAR⁺ T Cells.

Two of the three validation runs resulted in a preferential growth ofCD8⁺CAR⁺ T cells (mean 76%±22%) as compared to CD4⁺CAR⁺ T cells (mean16%±24%) (FIG. 7C) which was predicted by inclusion of IL-21 (Singh etal., 2011). Mean CD4/CD8 ratios for CAR⁺ T cells modulated during thecourse of the co-culture as there was a predominance of CD4⁺CAR⁺ T cellsat the start of the co-culture on aAPC (culture day 1, ratio=3.3)leading to equal amounts of CD4⁺CAR⁺ and CD8⁺CAR⁺ T cells at culture day14 (ratio=0.9), after which the ratio declined (culture day 21,ratio=0.4; culture day 28, ratio=0.3). The total CD4/CD8 ratio followeda similar trend and declined over time (Table 7). The CAR⁺ T cells atthe end of the propagation period (culture day 28) were activated (CD69expression, mean 43.6%; HLA-DR expression, mean 61.2%), capable ofcytolysis (Granzyme B expression, mean 92.3%) and expressed markers ofmemory/naïve T cells (CD62L expression, mean 45.6%; CD28 expression,mean 66.4%; CD27 expression, mean 77.5%, CD45RA expression, mean 99.7%).The inventors were not able to detect cell-surface markers ofexhaustion/senescence (PD-1 expression, mean 2.7%; CD57 expression, mean3.3%) (FIG. 7D). These data are consistent with the aAPC supporting theoutgrowth of a heterogeneous population of CAR⁺ T cells.

Redirected Specificity of CAR⁺ T Cells.

T cells generated from all three validation runs were able tospecifically lyse CD19⁺ tumor targets. An average of 57%±4% (mean±SD,range, 61.2% to 53.8%) Daudiβ₂m and 49%±7% (mean±SD, range, 41% to 54%)NALM-6 was lysed at an effector to target ratio of 20:1. CD19-specifickilling was demonstrated by average 6.2±2.6 (mean±SD)-fold higherkilling of CD19⁺ EL-4 (range, 4.2 to 9.2 fold) as compared to theCD19^(neg) parental EL-4 cells at effector/target ratio of 20:1 with a1.4±1 (mean±SD)-fold background CD19-specific lysis by CAR^(neg) (mockelectroporated) controls (FIGS. 8D, 14). This implies that the CAR inthe electroporated and propagated T cells redirected killing to CD19.

Lack of Unwanted Autonomous Proliferation by Genetically Modified andPropagated T Cells.

The inventors evaluated growth of CAW⁺ T cells in the presence/absenceof K562 aAPC and cytokines (IL-2 and IL-21) to rule out aberrant T-cellgrowth due to potential genotoxicity caused by SB transposition. At theend of 18 days of culture the inventors observed <2×10⁴ cells (average2,800, range 0.0-5.6×10³) in the genetically modified T cells receivingno cytokines and aAPC, while the control group receiving cytokines andaAPC numerically expanded to an average of 7.6×10⁷ cells, range 4.12×10⁷to 12.8×10⁷ (Table 8). These data indicate CAR⁺ T cells cannot sustainproliferation upon withdrawal of growth factors and antigenicstimulation.

TABLE 8 Lack of autonomous cell growth by genetically modified T cells.No. of T cells (day 18) Without With % fold- No. of cytokines cytokineschange Day of T cells and and of T Experiment culture^(a) seeded^(b)aAPC^(c) aAPC^(d) cells^(e) V1 28 2 × 10⁵ 0 12.80 × 10⁷  0.000% V2 28 2× 10⁵ 5.6 × 10³ 5.88 × 10⁷ 0.009% V3 28 2 × 10⁵ 2.8 × 10³ 4.12 × 10⁷0.007% ^(a)Days of culture when T cells were seeded ^(b)Total number ofT cells seeded in culture at the start of the experiments ^(c)Totalnumber of T cells counted in the absence of cytokines and aAPC ^(d)Total(inferred) number of T cells counted in the presence of cytokines andaAPC (positive control) ^(e)Perfect fold-change = [(c/b) ÷ (d/b)] * 100

TABLE 9 In-process testing for electroporated and propagated T cells.Expression Cell Surface CAR Expression Flow Cytometry Total CARExpression Western Blot Analysis Functionality Cytotoxicity ChromiumRelease Assay Persistence Memory/Naive Phenotype Flow Cytometry TelomereLength Flow-FISH Safety CAR Copy Number Q-PCR SB11 Detection PCR TCR VβRepertoire Flow Cytometry Karyotyping G-banding

Telomere Length in CAR⁺ T Cells.

Telomere length is an important measure of cellular differentiation andprogression to senescence. Therefore to evaluate the effect of SBtransposition and ex vivo numeric expansion of CAR⁺ T cells on telomerelength, the inventors compared telomere lengths from CAR⁺ T cells(culture day 28) to their respective matched unmanipulated controls(prior to electroporation) using Flow-FISH assay. Due to the generationof predominately CD8⁺ CAR⁺ T cells in V1 and V2 and CD4⁺ CAR⁺ T cells inV3, the inventors compared telomere lengths of CD8⁺ T cells for V1 andV2 and CD4⁺ T cells for V3 to CD8⁺ and CD4⁺ T cells, respectively, fromprior to propagation (FIG. 9A). The average telomere length of T cellsafter ex vivo numeric expansion (culture day 28, 8.93%±1.33%) wassimilar to that of unmanipulated control T cells (day 0, 7.77%±1.21%).These results indicate that electroporation and propagation of CAR⁺ Tcells does not result in erosion of telomere lengths.

Copy Number of CAR Transgene.

The copy number of integrated CD19RCD28 transgenes was determined usingCD19RCD28⁺ Jurkat cell clone #12 as reference and endogenous RNase P asa normalizer (Maiti et al., 2013). The average transgene copy per Tcells generated in the validation runs was 0.96±0.09 (range, 0.75 to1.07, FIG. 9B). These data indicate that SB transposition resulted inapproximately one integrated copy of CAR per T-cell genome.

TCR Vβ Usage.

The electroporation and propagation of T cells may lead to emergence ofoligoclonal or clonal population of T cells that could be indicative ofpreferential growth and thus an indicator of a genotoxic event.Therefore, the inventors evaluated TCR Vβ usage by flow cytometry as ameans to assess repertoire diversity. All 24 TCR Vβ families tested werepreserved in T cells after 28 days of co-culture on aAPC and similar tothe pre-electroporation repertoire. Further, the TCR Vβ families werepreserved upon prolong culture time (culture day 35, FIG. 9C). Thesedata suggest maintenance of a broad TCR diversity in cultured CAR⁺ Tcells and do not reveal an imbalance in the use of TCR sequences.

Lack of SB11 Transposase in CAR⁺ T Cells.

Continued expression of SB11 transposase may lead to remobilization ofthe integrated CAR transgene. Therefore, the inventors performed agenomic PCR to rule out illegitimate homologous recombination of the DNAplasmid coding for SB11 in CAR⁺ T cells. Within the limits of the assaythe inventors were unable to detect a band (˜1 kb) in PCR reactionscontaining DNA from CAR⁺ T cells cultured for 28 days and amplifiedusing SB11-specific primers (FIG. 9D). These data indicate a lack ofintegration of SB11 transposase in the electroporated and propagated Tcells.

Karyotype of CAR⁺ T Cells.

The integrity of chromosome structure was evaluated to rule out globalgenotoxicity associated with SB transposition. G-banding of CAR⁺ T cells(harvested 28 days after electroporation) from all the three validationruns revealed a normal (male) karyotype in all analyzed metaphasespreads (FIGS. 9E, 15).

Example 5 Targeting an Ancient Retrovirus Expressed in Cancers andInfections Using Adoptive T Cells Engineered to Express Chimeric AntigenReceptor—Methods

Immunohistochemistry.

Tissue microarray (#ME1004a, ME2082b, FDA998t) obtained from U.S. Biomax(Rockville, Md.) was hydrated with DiH₂O. Antigen retrieval usingcitrate buffer pH 6 without EDTA was performed. Slides were blocked with3% hydrogen peroxide (Biocare Medical, Concord, Calif.), avidin (BiocareMedical), biotin (Biocare Medical) and a polyvalent whole serum (BiocareMedical). Slides were incubated for 30 minute each with HERV-K mAb (0.6mg/ml) at 1:20 dilution followed by biotinylated anti-mouse IgG (BiocareMedical) and strepavidin-HRP (Sigma-Aldrich, St Louis, Mo.) andvisualized with a Mayer's Hematoxylin counterstain (Sigma-Aldrich).Similar staining procedures were performed on the slides with isotypecontrol mouse IgG2a (0.25 mg/ml) antibody (BD Pharmingen, FranklinLakes, N.J.).

Plasmids.

The scFv sequence (from mAb clone 6H5) against HERV-K envelope proteinwas codon optimized (CoOp) (Invitrogen, Carlsbad, Calif.) and clonedinto the SB transposon under control of the human elongation factor-1α(hEF-1α) promoter, flanked by SB inverted repeats forming_(CoOp)6H5CD28/pSBSO.

HERV-K antigen was expressed from SB plasmid containing bidirectionalpromoters hEF-1α and cytomegalovirus (CMV). The codon optimized fulllength antigen sequence with the viral transmembrane domain was clonedunder the control of the hEF-1α promoter and the neomycin gene wastranscribed under the control of the CMV promoter in a bi-directionalvector. The transposase (SB11) was expressed in cis from the plasmidpCMV-SB11 (Davies et al., 2010).

To generate the in vivo imaging SB plasmid for T cells, codon optimizedfirefly luciferase was fused to a myc tag and was expressed under thecontrol of the CMV promoter. A lentiviral vector encoding mKate-renillaluciferase under the control of the eEF1α promoter was used as animaging vector for melanoma tumor cells in vivo.

Cells Lines and their Propagation.

A375-mel and A888-mel were a kind gift from Dr. Lazio Radvanyi, TheUniversity of Texas MD Anderson Cancer Center (Houston, Tex.). A624-meland EL4 parental were obtained from American Type Culture Collection(Rockville, Md.). A375-SM (super-metastatic melanoma cell line) wasreceived from CCGS core facility at the University of Texas MD AndersonCancer Center (Houston, Tex.). All cell lines were cultured in RPMI(Thermo Scientific, Rockford, Ill.) with 10% FBS (Thermo Scientific) and5% glutamax (Gibco Life technologies, Grand Island, N.Y.). All celllines were verified by morphology, cell finger printing and/or flowcytometry. They were tested for Mycoplasma and conserved in researchcell banks.

Generation and Expansion of HERV-K-Specific CAR Expressing T Cells.

PBMCs from healthy donors were isolated by Ficoll-Paque density gradientcentrifugation (GE Healthcare Bio-Sciences, Piscataway, N.J.), andelectroporated with the HERV-K SB transposon and SB11 transposase usingthe Amaxa electroporation system. Briefly, 2×10⁷ PBMC cells were washedand incubated in complete RPMI supplemented with 10% fetal bovine serum(Thermo Scientific) and 1% glutamax (Gibco Life technologies) for 2 h.These cells were then resuspended in 100 μl of Amaxa Nucleofectorsolution (Human T-cell Kit), along with HERV-K CAR transposon(6H5CD28/pSBSO, 15 μg) and SB transposase (pCMV-SB11, 5 μg), transferredto a single cuvette, and electroporated using the U-14 program in theAmaxa electroporator (Lonza, USA). The electroporated T cells wereincubated for 4 h at 37° C. in complete phenol-free RPMI (ThermoScientific) after which a half-media change was performed.

K562 express endogenous HERV-K antigen and thus serve as aAPC topropagate HERV-K-specific CAR⁺ T cells. The electroporated T cellscultured in RPMI containing 10% FBS were supplemented with γ-irradiated(100 Gy) K562-aAPC at a 1:2 T cell:aAPC ratio. Irradiated aAPCs wereadded at the end of every week for T cell stimulation at the same ratio.Soluble IL-21 (eBioscience) and IL-2 (Chiron) cytokines weresupplemented at a concentration of 30 ng/ml and 50 U/ml, respectively,to complete RPMI media every other day in the culture afterelectroporation. Mock transfected No DNA control T cells grown in thepresence of OKT3 loaded K562 cells served as a negative control.CD19CAR⁺ T cells electroporated with _(CoOp)CD19CARCD28/p SB SO and SB11transposase were grown under the same culture conditions as in CAR⁺ Tcells and served as a non-specific CAR⁺ T cell control.

Every week the T cell cultures were monitored for the presence ofCD3^(neg)CD56⁺ cells and were depleted if the population exceeded 10% ofthe total population. This depletion usually occurred between 10 and 14days of initial co-culture with aAPCs. The depletion was carried outusing CD56 beads (Miltenyi Biotech Inc, Auburn, Calif.) on Automax(Miltenyi Biotech) using the positive selection “possel” according tomanufacturer's instruction.

T-cell viability was assessed based on trypan blue exclusion using aCellometer automated cell counter (Auto T4 Cell Counter, NexcelomBioscience, Lawrence, Mass.). The viability was analyzed using theprogram “PBMC_human_frozen” and “activated T cell,” duringelectroporation and co-culture period. The fold expansion (compared today 1) of total, CD3⁺, CD4⁺, CD8⁺ and CAR⁺ cells at the end of 7, 14,28, 32 days of co-culture for individual donors was calculated and theaverage of 3 donors were compared between the CD19CAR⁺ T cells and NoDNA control cells using a Student's t test.

Flow Cytometry.

All reagents were obtained from BD Biosciences (Franklin Lakes, N.J.)unless mentioned otherwise. One million cells were stained with antibodyconjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE),peridinin chlorophyll protein conjugated to cyanine dye (PerCPCy5.5), orallophycocyanin (APC). The antibodies used include anti-CD3 FITC (5 μl),anti-CD3 PerCPCy5.5 (2.5 μl), anti-CD3 PE (2 μl) anti-CD4 APC (2.5 λl),anti-CD8 PerCPCy5.5 (4 μl), anti-CD56 APC (2.5 μl), AnnexinV (5 μl),anti-CD32 FITC (5 μl), anti-CD45RA FITC (5 μl), anti-CD45RO APC (2.5μl), anti-Granzyme B FITC (5 μl), anti-CD62L APC (2.5 μl), anti-IFN-γAPC (2 μl), anti-CD27 PE (2 μl), anti-aPTCR FITC (5 μl), anti-γδTCR PE(2 μl), and anti-CCR7 PerCPCy5.5 (2.5 μl, Biolegend). FITC-conjugated (3μl, Invitrogen) and PE-conjugated (2.5 μl, Invitrogen) F(ab′)₂ fragmentof goat anti-human Fcγ was used to detect cell surface expression of theHERV-K-specific CAR. Blocking of nonspecific antibody binding wasachieved using FACS wash buffer (2% FBS and 0.1% sodium azide in PBS).Data acquisition was on a FACSCalibur (BD Biosciences) using CellQuestversion 3.3 (BD Biosciences). Analyses and calculation of medianfluorescence intensity (MFI) was undertaken using FlowJo version 7.5.5.

nCounter Analysis Digital Gene Expression System.

Difference in gene expression between HERV-K-specific CAR⁺ T cells andNo DNA control T cells were evaluated using the nCounter Analysis System(model no. NCT-SYST-120, NanoString Technologies; Geiss et al., 2008).Briefly, 10⁴ HERV-K-specific CAR⁺ T cells or No DNA T cells were lysedin RNeasy lysis buffer (RLT; 5 μl, Qiagen, Gaithersburg, Md.) and themRNA were hybridized with a reported code set and a capture code setcustom designed using the nCounter Gene Expression Assay Kit for 12 h at65° C. An nCounter prep station was used for the post-hybridiztionprocesses. Nanomir software was used to normalize the mRNA levels withan internal control. R-program, tree-view and clustal view were used tooutput the data with statistical analysis. The normalized results wereexpressed as the relative mRNA level. Ingenuity Pathway Analysis (IPA)(Ingenuity Systems, www.ingenuity.com) was performed on statisticallysignificant genes to understand their biological interaction based on adatabase derived from literature sources.

Integration Analysis.

Genomic DNA from HERV-K-specific CAR⁺ T cells was isolated using aQIAamp DNA mini kit (Qiagen) and real-time PCR was performed aspreviously described (Maiti et al., 2013). Genomic DNA from Jurkat cells(Clone #14) bearing a single integration of the CAR copy previouslydescribed was used as a positive control (Maiti et al., 2013). Theexperiment was done in triplicate with 100 ng of genomic DNA mixed with10 μl of TaqMan Gene Expression Master Mix (Applied Biosystems, FosterCity, Calif.), 1 μl (1× probe at 250 nM andlx primer at 900 nM) of 20×FAM-labeled CAR-specific TaqMan probe primer set specific for IgG4Fc[forward (5′-GAGGGCAACGTCTTTAGCTG-3′; SEQ ID NO: 15) and reverse(5′-GATGATGAAGGCCACTGTCA-3′; SEQ ID NO: 16) primers andcarboxyfluorescein (FAM)-labeled probe (5″-AGATGTTCTGGGTGCTGGTC-3′; SEQID NO: 17)] and 1 μl (1× primer at 900 nM and 1× probe at 250 nM) of 20×VIC labeled TaqMan RNaseP Probe Primer set (Applied Biosystems) in atotal reaction volume of 20 μl. The primer hybridization occurred at theIgGFc4 portion of the CAR. The amplification cycle included 2 minutes at50° C., 10 minutes at 95° C., and forty cycles of 15 seconds at 95° C.and 1 minute at 60° C. Detection was performed with a StepOnePlusReal-Time PCR System (Applied Biosystems). The autosomal RNaseP gene,present at 2 copies per diploid cell, was used as an endogenousreference for normalization (Jin et al., 2011). The ΔΔCT method (AppliedBiosystems, CA) was used to calculate the number of integrations withreference to RNaseP and Jurkat Clone as normalization controls.

Chromium Release Assay (CRA).

The CRA was performed as previously described (Maiti et al., 2013; Jinet al., 2011). Briefly, HERV-K^(+ve) targets were treated with ⁵¹Cr for2 h and incubated with day 35 HERV-K-specific CAR⁺ T-cells or No DNAcontrol T cells and the percentage of ⁵¹Cr release was calculated usingthe following formula:

${{\% \mspace{11mu}}^{51}{Cr}\mspace{14mu} {release}} = {\frac{{{Experimental}\mspace{14mu} {release}} - {{Background}\mspace{14mu} {release}}}{{{Maximum}\mspace{14mu} {release}} - {{Background}\mspace{14mu} {release}}} \times 100}$

Time-Lapse Bio-Imaging.

(A) To visualize CAR engagement with the antigen on the tumor cells,time lapse imaging was performed using a BioStation IM Cell-S1/Cell-S1-Psystem (Nikon, Melville, N.Y.). K562 parental cells were stained withanti-HERV-K APC antibody (1 μg) and the HERV-K-specific CAR⁺ T cellswere stained for CAR surface expression using FITC labeled F(ab′)2fragment of goat anti-human Fcγ antibody (5 μl, BD Biosciences). The Tcells and target cells were mixed at a ratio of 5:1 and plated incomplete RPMI culture medium on a T-35 mm glass bottom plate (FisherScientific, Hampton, N.H.). The cells were immediately imaged every 2minute at 37° C. for up to 8 h. Each image was recorded at 1600×1200pixels with a 20× objective, using phase-contrast, fluorescence channel2 to observe green HERV-K-specific CAR⁺ T cells, and fluorescencechannel 3 to observe red K562 cells with an exposure time of 1/125 and ⅕sec, respectively. CAR engagement with antigen was seen when theAPC-labeled antigen overlapped with the green-labeled CAR.

(B) To visualize and quantify the time taken for HERV-K specific CAR⁺ Tcells to kill a tumor cell, the tumor cells were plated withHERV-K-specific CAR⁺ T cells in a T-35 mm glass bottom plate withcomplete RPMI containing 1 ng/mg Sytox® (Invitrogen). Tumor cell deathwas recorded as the time when the tumor cell wall was punctured and thecells turned green using BiostationIM cell-S1-P system (Nikon). Theintensity of green fluorescence in each tumor cell was recorded usinglive cell imaging software (Nikon) over a period of 15 h.

Intracellular IFN-γ Release Assay.

HERV-K-specific CAR⁺ T cells were co-cultured with tumor cells at a 1:10ratio in a round-bottom 96-well plate with 200 μl of complete RPMIculture medium. Protein transport inhibitor (BD Golgi Plug containingBrefeldin A) was added in all wells to trap the IFN-γ inside the cell.The co-culture was incubated for 4 h at 37° C. and then stained forHERV-K-specific CAR expression for 20 min at 4° C. The cells were thenwashed, fixed, and permeabilized with 100 μl of Cytofix/Cytoperm buffer(catalogue no. 555028) for 20 min at 4° C. The permeabilized cells werethen stained for the cytokine with anti-IFN-γ APC conjugated antibody.The cells were washed and analyzed by FACSCalibur. PMA (phorbol 12myristate 13 acetate)- and ionomycin-(BD Biosciences) treated T cellswere used as positive controls for this assay. Similar assays wereperformed with No DNA control T cells.

Development of HERV-K^(+ve) EL4 Cell Line.

Two million EL4 cells were suspended in AMAXA mouse T cell nuclofectorsolution (Amaxa, USA) with HERV-K antigen-expressing SB transposon andSB11 transposase (2 μg of total DNA) to a final volume of 100 ml. Thissuspension was transferred to a single cuvette and electroporated usingthe C-09 program in Amexa electroporator. The cells were incubated for 4h at 37° C. in electroporation media supplied with the kit supplementedwith 10% FBS (Thermo Scientific Pierce). The cells were then transferredto DMEM, 10% FBS (Thermo Scientific Pierce) and 5% glutamax (Gibco LifeTechnologies). These cells were then grown in the presence of 0.8 mg/mlof G418 and neomycin-resistant HERVK^(+ve) EL4 cells were sorted usingHERV-K antibody and grown to obtain a pure population of cells.

shRNA-Mediated HERV-K Knockdown in A888-Mel Cells.

A888-mel cells were grown to 90% confluence in a 6-well plate. The mediawas later replaced using 100 μl of either HERV-K-specific shRNA orscrambled shRNA lentivirus and polybrene (5 μg/ml) and transduced for 4h at 37° C. and then replaced with regular RPMI media. The cells werethen sorted based on GFP expression and grown. ScrambledshRNA-transduced A888-mel cells were used as control. An immunoblotassay of the cell line lysates was performed to determine the extent ofHERV-K knockdown. The 6H5 HERV-K antibody was used to detect the HERV-Kantigen expression on A888-mel cells with HERV-K shRNA or scrambledshRNA or parental cells. Ten million cells were lysed with RIPA buffercontaining protease inhibitor (Roche Applied Science, San Francisco,Calif.). BCA assay was performed to detect protein concentration (ThermoScientific Pierce). A 4%-20% gradient gel (Biorad, Hercules, Calif.) wasused to run 10 μg of protein boiled in SDS loading buffer. The proteinwas then transferred to a nitrocellulose membrane and blocked with 5%milk in PBST and incubated with 6H5 HERV-K antibody (mg/ml). Binding wasdetected by goat anti-mouse Fc-HRP (Sigma-Aldrich) and developed usingECL Westfemto™ substrate (Thermo Scientific Pierce). The blot was imagedusing Versa doc Quantityone™ software (Biorad) and blot quantified usingImage J software.

In Vivo Analysis.

Metastatic Melanoma Model:

5-week-old female NOD.Cg-Prkdc_(scid)Il2rg_(tmIwjl)/SzJ (NSG) mice(Jackson Laboratories, Bar Harbor, Me.) were intravenously injected with10⁶ A375-SM cells on Day 0. A375-SM cells were previously stablytransduced with mKate-rRLuc and cell sorted for homogenous population.Mice in the treatment cohorts (n=7) started receiving 2×10⁶HERV-K-specific CAR-FfLuc T cells starting on Days 7, 14 and 21. IL-2(600 U; eBioscience) was injected intraperitonealy (i.p.) three times aweek during the treatment period. One cohort of mice (n=6) bearing thetumor received no treatment while a control group of mice (n=3) withouttumor received a similar number of CAR⁺ T cells as in treatment group.

Flux Quantification:

Bioluminescence imaging (BLI) was performed every week to image thetumor and T cell activity in vivo. Mice were anesthetized and placed inanterior-posterior position for BLI using a Xeno IVIS 100 series system(Caliper Life Sciences) as previously descreibed (Singh et al., 2007).To image the HERV-K-specific CAR-ffLuc T cell activity, 150 μl (200μg/mouse) of D-Luciferin potassium salt (Caliper Life Sciences) wasinjected intraperitoneal (i.p.). Ten minutes after injection emittedphotons were quantified using the Living Image 2.50.1 (Caliper LifeSciences) program. To image the tumor cell activity, 100 μl of Endurin(Promega, Fitchburg, Wash.) was injected i.p. Twenty minutes afterinjection the tumor activity was quantified similar to ffLuc. Unpairedstudent's t-tests were performed to establish statistical significanceof the flux.

Statistical Analysis.

For analyzing statistical differences in antigen expression betweenvarious grades and stages of melanoma and normal tissue, student'st-tests and ANOVA were used. For analyzing the differences betweencontrol versus HERV-K-specific CAR⁺ T cell expansion, phenotypeanalysis, and functional assays, student's t-tests, means, standarddeviations, and 95% confidence intervals (CIs) were calculated. For themetastatic melanoma model, in vivo, student's t-tests were used toanalyze the significance in the flux data between the tumor andtreatment group. All statistical tests were two-sided and performedusing Graph Pad Prism software (GraphPad Software Inc, San Diego,Calif.). All P values less than 0.05 were considered statisticallysignificant.

Example 6 Targeting an Ancient Retrovirus Expressed in Cancers andInfections Using Adoptive T Cells Engineered to Express Chimeric AntigenReceptor—Results

Expression of HERV-K in Melanoma Patient Samples.

To elucidate the physiological relevance of HERV-K during melanomainvasion and metastasis in vivo, the inventors sought to assess theexpression of the tumor antigen HERV-K envelope protein in biopsiesobtained from patients with various stages of melanoma. Malignant tumortissues from two hundred sixty-eight patients and benign skin and breasttissue from forty patients were analyzed using IHC. The tumor tissuestaining was graded based on the percentage of tumor cells positive forHERV-K and the intensity of staining and their product was calculated toobtain the H-index. Tumor tissues showed varied level of stainingintensity and were scored 0, 1, 2 or 3 (FIG. 16A) when compared to theisotype control staining on the same tissue. The antigen was expressedon the cells either in a punctuate form along the cell surface as shownwith a solid arrow or as diffuse cytoplasmic staining as shown withdotted arrow (FIG. 16B). This may suggest the circulation andaccumulation of the antigen onto the cell surface for the purpose ofshedding the viral protein (REF). Tumor cells express significantlyhigher H-index of HERV-K antigen compared to benign tumor (FIG. 16C).Though there is no difference in H-index between the malignant andmetastatic tumor, a significant difference can be seen between thetumors in stage I and II compared to tumors in stage III and IV (FIGS.16D, E). In order to further show the specificity of HERV-K expressionon tumor cells and not on normal cells, tissue from thirty-three typesof normal organ each obtained from three normal donors were analyzed. Nosignificant expression of HERV-K was observed in any of the normal organtissues (FIG. 22). These findings suggest that HERV-K up-regulationspecifically in tumor cells can serve as a unique target marker forimmunotherapy.

Propagation and Characterization of HERV-K-Specific CAR⁺ T Cells.

The HERV-K env-specific monoclonal antibody (mAb) was developed in mouseand the 6H5 mAb clone was found most sensitive in detecting antigen invitro (Wang-Johanning et al., 2003). The scFv sequence of the 6H5 mAbclone was used to construct the CAR. The scFv cassette was fused toIgG4Fc region by a flexible linker, followed by the CD28 transmembraneand CD28 and CD3z intracellular domains. This was then cloned into theSB transposon vector (FIG. 17A).

To generate CAW⁺ T cells specific to HERV-K env antigen, the inventorselectroporated peripheral blood mononuclear cells (PBMCs) with SBtransposon along with SB11 transposase and propagated the cells onendogenously-derived HERV-K^(+ve) K562 aAPC. To selectively propagate Tcells with stable expression of CAR, these aAPC, which endogenouslyexpress the HERV-K antigen, were genetically modified to co-expressdesired T-cell co-stimulatory molecules CD86, 4-1BBL, and membrane boundIL-15 (co-expressed with enhanced green fluorescent protein, EGFP)(Singh et al., 2008). PBMCs without any transposon electroporated, grownon OKT3-loaded aAPCs under the same culture conditions served as anegative No DNA control.

The expression of CAR was detected using a polyclonal Fc antibodyspecific for the IgG4Fc region. The CAR⁺ T cells were stained with Fcantibody every seventh day before supplementing the culture withirradiated aAPCs. The flow data revealed that 95% of T cells express CARon its surface, which could be detected for up to 35 days of culture(FIG. 17B). No significant difference was seen in the growth kinetics ofHERV-K-specific CAR⁺ T cells when compared to No DNA control cells, andall the HERV-K-specific CAR⁺ T cells were CD3⁺ T cells by day 14 ofculture (FIGS. 17C, D). The percent average number of CAR⁺ CD4 increaseswhile CAR⁺ CD8 cells decreases along the culture period (FIG. 23A).Real-time PCR analysis on the genomic DNA of HERV-K-specific CAR⁺ Tcells compared to No DNA control cells showed that there are less thantwo integration of the CAR in the CAR⁺ T cell genome (FIG. 17D). Thecultured CAR⁺ T cells have an effector memory phenotype, which includesCD3⁺ CD56⁺CD45RO⁺ TCRαβ⁺CD27^(neg)CCR7^(neg) cells with substantiallytic potential observed by Granzyme B levels (FIG. 17F).

The mRNA levels of HERV-K-specific CAR⁺ T cells from three normal donorswere measured and compared to mRNA levels from No DNA control T cells onDay 28 of culture using nCounter analysis. HERV-K-specific CAR⁺ T cellshad significantly higher levels of chemoattractants, transcriptionalregulators and activators. Increased levels of Perforinl and Granzyme Hin CAR⁺ T cells shows the higher lytic potential of these cells (FIG.23B). Ingenuity Pathway Analysis (IPA) (p<0.05) suggested several ofthese upregulated genes in HERV-K-specific CAR⁺ T cells were involved inNF-κB activation. The chemoattractant and the cytokines were involved inIL-10, IFN-γ and IL-12 regulation (FIG. 23C). These data strengthen theprevious observation that HERV-K-specific CAR⁺ T cells have a centraleffector phenotype.

Characterization of HERV-K CAR⁺ T Cell Functionality.

The antigen expression on melanoma cell lines, such as A888, A375,A375-SM, A624 were analyzed using the monoclonal 6H5 antibody directedagainst the HERV-K env protein (FIG. 18A). The HERV-K antigen expressionon melanoma cells were compared with the isotype control (mouse IgG2a).In order to analyze the functionality of HERV-K-specific CAR⁺ T cells,HERV-K⁺ tumor cells were pulsed with radioactive chromium and culturedwith varying ratios of HERV-K-specific CAR⁺ T cells. CRA was performedwith no DNA control as a negative control. Significantly higher levelsof HERV-K⁺ tumor cell lysis were observed with HERV-K-specific CAR⁺ Tcells when compared to no DNA control T cells (FIG. 18B). An unrelatedCAR, such as CD19-specific CAR⁺ T cells were also used to perform CRAand basal levels of non-specific killing were observed with HERV-Kantigen positive tumor cells when compared to CD19 antigen positivetumor cells, such as EL-4 cells bearing CD19 antigen (FIG. 24A).

In order to further prove the functionality of these HERV-K-specificCAR⁺ T cells, a four-hour IFN-γ release was performed. Melanoma tumortargets were co-cultured with HERV-K-specific CAR⁺ T cells or No DNAcontrol T cells at a 1:10 ratio and intracellular cytokine levels wereanalyzed using flow cytometry. T cells cultured with PMA-Ionomycinserved as a positive control. The HERV-K-specific CAR⁺ T cells showedhigher levels of IFN-γ release compared to the no DNA control T cells(FIG. 18C) and this result correlates with the CRA.

Specificity of HERV-K-Specific CAR⁺ T Cells.

To show the specificity of the HER-K-specific CAR, HERV-K^(neg) EL-4cells were electroporated with bi-directional SB vector encoding HERV-Kantigen under the hEF-1α promoter and neomycin resistance under the CMVpromoter (FIG. 25A). The EL-4 HERV-K^(+ve) cells were then single cellsorted and grown in the presence of mammalian selection marker,neomycin, to obtain a pure HERV-K env-expressing population.Interestingly, these cells lost the HERV-K antigen expression within tendays of culture due post-translational modification and proteolyticcleavage. A four-hour CRA was performed on these EL-4 HERV-K^(+ve) cellswithin ten days of sorting. Chromium pulsed EL-4 HERV-K^(+ve) cells andHERV-K^(neg) EL4 parental cells were co-cultured with varyingconcentrations of HERV-K-specific CAW⁺ T cells and graded tumor-specificlysis was observed in an antigen-specific manner (FIG. 19B).

To further prove the specificity, HERV-K env antigen was knocked down inA888 melanoma cells using shRNA lentivirus. An immunoblot analysisshowed about 50% knockdown in the HERV-K protein level compared to theA888 parent and control scrambled shRNA (FIG. 19C). A CRA ofHERV-K-specific CAR⁺ T cells with the HERV-K knockdown cells, A888parental, and A888-with scrambled shRNA showed significant reduction inkilling with HERV-K knockdown compared to the parent and control tumorcells (FIG. 19D).

Time lapse imaging was performed to visualize and quantify the timetaken for CAR⁺ T cells to kill the tumor target and the number of tumorcells killed over a period of 15 h. The tumor cells and T cells werecultured at a ratio of 1:5 in the presence of Sytox®, which turns cellsgreen when the cell membrane is damaged. As the melanoma target A888 andA375 cells were killed by the HERV-K-specific CAR⁺ T cells, a spike ingreen florescence was reported around 5 h, which gradually increasedover a 15 h time period. No killing was observed with HERVK-K^(neg)HEK293 parental targets. About 30% of A888 cells and 35% of A375 cellswere killed by CAR⁺ T cells over a period of 15 h. To visualize theantigen-specific CAR engagement, K562 cells were stained positive forHERV-K antigen in red and HERV-K-specific CAR was stained for Fc ingreen. The overlap of CAR and antigen was seen over a period of 5 hafter which the antibody-bound antigen and CAR were internalized by thecells (FIG. 26A).

Tumor Killing Ability of HERV-K-Specific CAR⁺ T Cells In Vivo.

PBMCs were double electroporated with SB vectors encodingHERV-K-specific CAR and myc-FFLuc with SB11 transpsosase. The SB vectorwith the myc-ffLuc gene has a neomycin resistance gene attached througha linker (FIG. 27A). These HERV-K-specific CAR-ffLuc⁺ T cells hadsimilar growth kinetics as the HERV-K-specific-CAR⁺ T cells and theirtumor killing ability and specificity was comparable (FIGS. 27B, C).

A metastatic melanoma model in which the A375-super metastatic (A375-SM)cell line was infused though the tail vein of NSG mice was developed.These cells engrafted in the lungs and metastasized to the liver. Inorder to visualize the reduction in tumor mass non-invasively, the tumorcells were transduced with a lentiviral vector bearing the mKate-rRLucgene and sorted using mKate marker to obtain a pure population of thecells (FIG. 21A). After a week of tumor cell engraftment, 20 millionCAR⁺ T cells were infused intravenously on days 8, 15, and 22 days alongwith IL-2 (i.p.) twice a week for three weeks. Bioluminescence imaging(BLI) was used to assess the rRLuc activity in each group of mice. Themouse group with tumor cells alone had significantly higher luciferaseactivity by day 25 compared to the mouse group with tumor cells thatreceived the HERV-K CAR⁺ T cells (FIGS. 21A, B). Also mice with tumoralone became moribund by day 28 due to the high metastasis of the tumorto the liver while the mouse group receiving the CAR⁺ T cells exhibiteda healthy appetite and activity. Ex vivo imaging of mKate on tumor cellsshowed that the tumor alone mice had substantially more tumor colonieson the liver than the treatment group (FIG. 21C). Pathologicalexamination proved this observation where the tumor group hadsignificantly higher metastatic colonies in the liver compared to thetreatment group suggesting the role of HERV-K CAR⁺ T cells in reducingtumor growth and metastasis (FIG. 21D).

Discussion.

These results show that HERV-K-specific CAR⁺ T cells were able tosuccessfully target a viral glycoprotein and kill HERV-K⁺ tumor cells inan antigen-specific manner in vitro and reduce the melanoma tumor growthand metastasis in vivo.

HERV-K env is markedly up-regulated during melanoma metastasis andexclusively present on tumor cells and not on adjacent normalmelanocytes. In melanoma, HERV-K env protein is associated with MEK-ERKand p16INK4A-CDK4 pathway activation relating to tumor progression (Liet al., 2010). HERV-K molecular mimicry also results in reducedglutathione peroxidase levels resulting in increased reactive oxygenspecies in tissue leading to melanomogenesis (Krone et al., 2005).Abnormal levels of HERV-K env expression appear to be a trigger factorinvolved in melanoma onset resulting in morphological and cellularmodifications resulting in tumor progression (Serafino et al., 2009).Modulation of viral transcription of HERV-K resulted in increasedinflammation and morphological differentiation in melanoma cells(Sciamanna et al., 2005). The tissue microarray represents increasedlevels of antigen expression correlating with melanoma progression whilethe normal human tissue has a basal level or an absence of HERV-Kexpression.

The immunogenic nature of melanoma makes it an attractive model andtarget to develop potent T cell-based therapy. One of the majorlimitations on T cell-based therapy is the HLA-based restriction on TCR,which limits antigen recognition (Garrido et al., 1997. Geneticallymodifying T cells to express tumor antigen specific receptor circumventsthis restriction and confers non-HLA-based antigen recognition (Gross etal., 1989). The T cell modifications can be brought about by usingeither viral or non-viral vectors. The Sleeping Beauty vector is anon-viral approach to introduce genes into T cells. One of the mainadvantages of the Sleeping Beauty system over other viral transductionmethods is that it is genetically safe, efficient, and less expensivethan retroviral transduction (Maiti et al., 2013). Culturing these CAR⁺T cells with IL-2 and IL-21 yields enough cells for infusion purposes.This has led to preliminary clinical trials using CD19 CAR⁺ T cellsagainst B-lineage malignancies. HERV-K-specific CAR⁺ T cells were grownin a similar manner to the clinical-grade CD19 CAR⁺ T cells. TheseHERV-K-specific CAR⁺ T cells were mainly of an effector memoryphenotype, which are well adapted to target and kill tumor cells.

Multiple factors, such as cytokines, hormones and chemicals, are knownto regulate HERV-K levels during cancer (Taruscio and Mantovani, 2004).The HERV-K env antigen is known to circulate between the cell membraneand cytoplasm and also bud off from the cell surface in certain tumorconditions. Hence, using adoptive T cell therapy can be a favorabletreatment strategy that only requires transient antigen expression onthe cell surface.

HERV-K env antigen is also found associated with breast cancer, ovariancancer, prostate cancer, lymphoma, teratocarcinoma, autoimmune diseases,such as multiple sclerosis, and infectious diseases, such as HIV(Wang-Johanning et al., 2003; Contreras-Galindo et al., 2008; Jones etal., 2012; Ono, 1986; Wang-Johanning et al., 2007). Thus, targeting theenv antigen using adoptive T cell therapy can be a treatment option formultiple disease conditions. Consistent with this hypothesis, HERV-K envcan be successfully and selectively targeted by HERV-K CAR⁺ T cells.Infusing these genetically modified CAR⁺ T cells in vivo was associatedwith tumor regression and reduced metastasis showing the antitumoractivity of these cells.

Example 7 Using Membrane-Bound Cytokine(s) to Generate T Cells withLong-Lived In Vivo Potential for Use in Immunotherapy of MinimalResidual Disease

Generation and Expression of mIL15.

The mIL15 construct (FIG. 28) fuses the IL-15 cDNA sequence(NM_000585.4) to the full length IL-15Rα (NM_002189.3) with aserine-glycine linker. The signal peptides for IL-15 and IL-15Rα wereomitted and the IgE signal peptide (gb|AAB59424.1) was used for themIL15 construct. This construct will produce an IL-15 that ismembrane-bound, but also presented in the context of IL-15Rα which isakin to the trans-presentation model described above. The DNA plasmidswere synthesized by GeneArt (Regensburg, Germany) and subsequentlysubcloned into a Sleeping Beauty plasmid (a non-viral gene transfermethod). Primary human T cells were co-electroporated with a CAR plasmid(specific for CD19), with or without the mIL15 plasmid, and the SB-11transposon. Propagation and expansion of the genetically modified Tcells was achieved by weekly stimulation a CD19⁺ K562 artificial antigenpresenting cell (aAPC) variant expressing 41BBL and CD86 co-stimulatorymolecules. The CAR used is a 2nd generation CAR containing CD3ζ and CD28signaling cytoplasmic domains. The presence of CD19 on the aAPC allowsfor the selective outgrowth of antigen-specific T cells while thecostimulatory molecules improve in vitro expansion. The mIL15 moleculecan be stably co-expressed with the CAR by the modified T cells and theco-expressing T cells represent the bulk of population (FIG. 29).Additionally, total CAR-expression in the modified T cells reachesgreater than 90% for the mIL15-CAR-modified T cells (FIG. 29).

Functionality of mIL15.

IL-15 receptor complex signaling primarily induces phosphorylation ofsignal transducer and activator of transcription 5. By looking atphosphorylated STAT5 (pSTAT5) using phosflow, it can be determinedwhether mIL15 is capable of inducing the cytokine signaling pathway. ThepSTAT5 levels were elevated in CAR⁺ T cells having had cytokinesupplementation with these levels abrogated under serum and cytokinestarvation. Under starvation conditions, pSTAT5 levels in mIL15⁺CAR⁺ Tcells were maintained (FIG. 30). These data demonstrate that mIL15 isfunctional and activates the pSTAT5 portion of the cytokine signalingpathway.

Propagation of Clinically Significant Numbers of mIL15⁺CAR⁺ T Cells.

In redirecting T cell specificity with the CAR, the focus in ex vivoexpansion is on driving the CAR⁺ T cell population, in this case CARwith or without mIL15. The standard CAR⁺ T cells are grown with solubleIL-2 and IL-21 while mIL15⁺CAR⁺ T cells were given soluble IL-21 tocapitalize on a reported synergy between IL-15 and IL-21. The mIL15⁺CAR⁺T cells supplemented with IL-21 demonstrated comparable expansion to thestandard CAR⁺ T cells, as well as the control CAR⁺ T cells given IL-15and IL-21 (P=0.53, 2-way ANOVA; FIG. 31). The ex vivo expansion ofmIL15⁺CAR⁺ T cells produces clinically significant numbers of cells.

Assessing the Phenotype and Functionality of mIL15⁺CAR⁺ T Cells.

The phenotype of the ex vivo expanded mIL15⁺CAR⁺ T cells are largelysimilar to the CAR⁺ T cells except for IL-7Ra expression. The generalphenotype of the cells, which at this time point represents the infusionproduct, are predominantly CD8⁺ (cytotoxic) T cells with moderate tohigh expression of the activation markers CD45RO and CD25. There werevariable low to moderate levels of the expression of T cellmemory-associated markers (CD62L, CCR7, CD27, and CD28) (FIG. 32A) forboth T cell groups. After ex vivo expansion of the modified T cells, itis necessary for the T cells to retain their redirected T cellspecificity and lytic function. A chromium release assay was conductedto assess the function of the T cells in the expansion product. CD19⁺and CD19⁻ EL4 targets were plated with the modified T cells at varyingeffector to target ratios. Specific lysis of CD19⁺ tumor targets wasdemonstrated across all effector to target ratios by both mIL15⁺CAR⁺ Tcells and CAR⁺ T cells (P<0.001, 2-way ANOVA, n=3) and did not differfrom one another (P >0.05, 2-way ANOVA). CD19⁺ target lysis bymIL15⁺CAR⁺ T cells was specific and significantly different frombackground lysis of CD19⁻ targets (P<0.001, 2-way ANOVA) (FIG. 32B). ThemIL15⁺CAR⁺ T cells retained their redirected specificity and lyticcapacity.

Specific mIL15⁺CAR⁺ T Cell Subsets Persist Long-Term In Vitro and RemainFunctional.

To assess long-term in vitro persistence, four aAPC stimulation expandedmIL15^(+/−)CAR⁺ T cells were cultured long-term without further antigenre-stimulation. CAR⁺ T cells received IL-2, IL-15, or no cytokinesupplementation while mIL15⁺CAR⁺ T cells did not receive exogenouscytokines. As anticipated, CAR⁺ T cells receiving no cytokinesupplementation did not persist. The mIL15⁺CAR⁺ T cells as well as CAR⁺T cells receiving IL-2 or IL-15 had significantly greater relative foldexpansion than the unsupplemented CAR⁺ T cells (P<0.0001, repeatedmeasures ANOVA, n=3; FIG. 33A). The maintenance of mIL15⁺CAR⁺ T cellrelative expansion near zero also suggests that these modified cells arenot growing in an unrestricted manner. To be a benefit for the clinicalapplication, CAR⁺ T cells must remain responsive to antigen. Hence,these T cells at 75+days from antigen encounter were challenged with: notarget, CD19⁻ EL-4, CD19⁺ EL-4, CD19⁺ Nalm-6 (a human leukemia cellline), or lymphocyte activating cocktail (LAC) and interferon γ (IFNg)production was assessed by intracellular cytokine staining after 6 hoursincubation with the targets. Similarly to the CAR⁺ T cells receivingeither IL-2 or IL-15, the mIL15⁺CAR⁺ T cells also produced IFNg inresponse to CD19⁺ targets and the LAC (FIG. 33B). In another assay,these 75-day withdrawal T cells were stimulated with aAPC andsupplemented with IL-21. Thus, CAR⁺ T cells cultured with IL-2 now hadIL-2 and IL-21 provided, the IL-15 culture T cells then received IL-15and IL-21, and the mIL15⁺CAR⁺ T cells were supplemented with IL-21 only.T cell viability was assessed via Annexin V staining eight days afterthe aAPC stimulation. The mIL15⁺CAR⁺ T cells were shown to have thegreatest live cell population (Annexin V^(neg)) (FIG. 33C) and indicatesa resistance to activation induced cell death.

Persisting mIL15⁺CAR⁺ T Cells Possess Traits of Less Differentiated TCells.

In characterizing the long-term persisting mIL15⁺CAR⁺ T cells, theinventors hypothesized that persisting mIL15⁺CAR⁺ T cells would exhibitcharacteristics associated with less differentiated T cell subsets asthese cell subsets are known for their long-lived potential. With theconstitutive presence, and thus possible constitutive signaling, duringthe ex vivo culture of the mIL15⁺CAR⁺ T cells, the inventors assessed ifthese T cells had a molecular programming for a less differentiatedstate that would yield cells with a persistence advantage. Analysis ofmultiplexed digital gene profiling using the nCounter Analysis Systemwas performed on CAR⁺ and the mIL15⁺CAR⁺ T cells from stimulation 4(withdrawal assay T cell input). Analysis of normalized mRNA counts useda negative binomial distribution-based statistical program (Lohse etal., Nucleic Acids Res. (2012) 40, W622-7). Genes were consideredsignificantly differentially expressed if greater than two-folddifferential mRNA counts, P<0.05, FDR q<0.05. Only five genes wereconsidered differentially expressed using these criteria, thusindicating there is no culture or molecular programming advantageafforded by mIL15.

The inventors then characterized the long-term persisting mIL15⁺CAR⁺ Tcells. First, their phenotype was assessed using CD45RA and CCR7 markersto phenotypically describe their differentiation state. These markerscharacterize the differentiation state of T cells asCD45RA⁺CCR7⁺<CD45RA⁻CCR7⁺<CD45RA⁻CCR7⁻<CD45RA⁺CCR7⁻ representing cellsfrom the least to most differentiated state. In comparing the 75-daywithdrawal mIL15⁺CAR⁺ T cells to their counterparts at the initiation ofthe experiment (the Stim 4 mIL15⁺CAR⁺ T cells), it is observed that thepersisting mIL15⁺CAR⁺ T cell culture has an increased proportions ofCD45RA⁺CCR7⁺ and CD45RA⁺CCR7⁻ T cell subsets (***P<0.001, 2-way repeatedmeasures ANOVA, n=7; FIG. 34). CCR7 expression was significantlyenhanced in the withdrawal mIL15⁺CAR⁺ T cells relative to CAR T cells.Viability of CCR7^(neg) and CCR7⁺ subsets was assessed by Annexin Vstaining of mIL15⁺CAR⁺ T cells, CAR⁺ T cells receiving IL-2 (50 U/ml),and CAR⁺ T cells receiving soluble IL-15 (5 ng/ml) after antigenwithdrawal. It was found that irrespective of the type of cytokinestimulation (IL-2, IL-15 complex, or mIL15), CCR7^(neg) T cells showedequal frequencies of live cells. In contrast, CCR7⁺ T cells exposed tomIL15 had significantly higher viability than the CAR T cells receivingIL-2 or IL-15 complex (both P<0.05; FIG. 34). These data suggest thatmIL15 is sufficient to support the CCR7⁺ phenotype which thuscontributes to maintaining the less differentiated CD45RA⁺CCR7⁺ T cellsubset. The capacity for the mIL15⁺CAR⁺ T cells to promote thepersistence of less differentiated T cells is a desired phenotype foradoptive therapy and appears to corroborate other studies reporting thatlong-lived T cells subsets possess a less differentiated phenotype.Additionally, the survival of a highly differentiated subset was alsoobserved, possibly supported by constitutive IL-15 signaling.

Long-term persisting mIL15⁺CAR⁺ T cells display some molecular markersassociated with less differentiated T cell subsets. The T cells wereanalyzed for their gene expression patterns using the nCounter AnalysisSystem and a heirarchically clustered heat map of differentiallyexpressed genes between mIL15⁺CAR⁺ T cells from stimulation 4 and thosesurviving to day 75 of withdrawal was produced (>2-fold cutoff, P<0.05,FDR q<0.05). These studies identified 108 significantly differentiallyexpressed genes (>2-fold cutoff, P<0.05, FDR q<0.05). Gene Ontologyclassification was assessed using DAVID functional annotation. Thefunctional classification of the differentially expressed genes can begrouped into broad categories: T cell activation, differentiation,proliferation, and apoptosis. Namely, there were greater numbers ofgenes in mIL15⁺CAR⁺ T cells that were down-regulated in the positiveregulation of differentiation, regulation of apoptosis, and induction ofapoptosis. Greater numbers of genes in mIL15⁺CAR⁺ T cells wereup-regulated in the negative regulation of differentiation and the Wntsignaling pathway (FIG. 36). This suggests that the molecular signatureof persisting mIL15⁺CAR⁺ T cells is less differentiated than mIL15⁺CAR⁺T cells at stimulation 4 (T cells at experiment initiation).

Expression of selected genes was validated by flow cytometry. Expressionof transcription factors associated with a less differentiated state(Tcf-7) and acquisition of effector function/differentiated state(Blimp-1 and Tbet) indicate that persisting mIL15⁺CAR⁺ T cells exhibit atranscription factor balance associated with less differentiated cells.This is characterized by greater expression of Tcf-7 and lowerexpression of Blimp-1 and Tbet (FIG. 37). Assessment of the cell surfacemarkers IL-7Ra and CCR7 was done as they are characteristicallyexpressed by less differentiated T cell subsets. The persistingmIL15⁺CAR⁺ T cells have increased expression of these markers associatedwith long-lived T cell subsets (FIG. 38). One additional measure fordistinguishing the level of T cell differentiation is by the capacity ofless differentiated T cells to produce IL-2. The mIL15⁺CAR⁺ T cells fromeither stimulation 4 or the 75-day withdrawal cultures were mock-treatedor treated with LAC for 6 hours and then assessed for IL-2 production byintracellular cytokine staining. The stimulation 4 T cells were unableto produce IL-2 whereas the persisting 75-day withdrawal T cellsacquired the capability to produce IL-2 (FIG. 39). These resultscollectively suggest that while there is no identifiable differencebetween ex vivo expanded CAR⁺ T cells or mIL15⁺CAR⁺ T cells, theresulting long-term persisting mIL15⁺CAR⁺ T cells exhibitcharacteristics associated with less-differentiated T cells subsetswhich demonstrate long-term survival in vivo.

In Vivo Persistence and Anti-Tumor Efficacy of mIL15⁺CAR⁺ T Cells in aHigh Tumor Burden Model.

To assess in vivo persistence, mIL15⁺CAR⁺ T cells and CAR⁺ T cells wereco-modified to express firefly luciferase (ffLuc) to enable longitudinalmonitoring of T cells in vivo using bioluminescence imaging (BLI). Thesemodified T cells were adoptively transferred, using one T cell infusionof 20×10⁶ CAR⁺ T cells, into NSG mice bearing disseminated Nalm-6 (CD19)malignancy with a no treatment control group. After 14 days the micewere sacrificed. While CAR⁺ T cells were not found to persist, themIL15⁺CAR⁺ T cells were observed by BLI to persist throughout the 11 dayimaging period (FIG. 40B). Bone marrow, spleen, and peripheral blood,were harvested and assessed by flow cytometry for the presence of humanT cells using human CD3 as a marker and gating out murine lymphocytes.Mice infused with the mIL15⁺CAR⁺ T cells had significant CD3⁺ T cellsdetected in the bone marrow (0.49-2.17%, P=0.0001, unpaired t-test,n=5), spleen (1.15-12.38%, P<0.0001, unpaired t-test, n=5) andperipheral blood (58.39-92.60%, P<0.0001, unpaired t-test, n=5) (FIG.40C). There were no CD3⁺ cells detected in samples from the CAR⁺ T celltreated group (FIG. 40C) and the no treatment group (tumor only) in anyof the assessed tissues. In this model, mIL15⁺CAR⁺ T cells demonstratedtumor control in the peripheral blood (FIG. 40D), but complete tumorclearance was not observed. These data indicate that despite theprevalence of tumor antigen, CAR⁺ T cells had insufficient in vivopersistence whereas mIL15⁺CAR⁺ T cells were present at significantlevels throughout the body.

In Vivo Persistence and Anti-Tumor Efficacy of mIL15⁺CAR⁺ T Cells in aLow Tumor Burden Model.

The inventors next assessed T cell engraftment and anti-tumor activityin a preventative model with a low tumor burden. The modified T cells(mIL15^(+/−)CAR⁺ffLuc⁺) were infused into the NSG mice with no exogenouscytokines and allowed to engraft for six days before an infusion ofrenilla luciferase (rLuc)-modified Nalm-6. The mice underwent BLI overthe course of 30 days. In this preventative model, the mIL15⁺CAR⁺ Tcells were observed to persist throughout the duration of the experimentand prevented tumor engraftment, which was a significant anti-tumoreffect compared to the CAR⁺ T cell and no T cell treatment groups(P<0.0001, one-way ANOVA, n=4-5) (FIG. 41C-D). Analysis of the organsand peripheral blood by flow cytometry detected human CD3⁺ T cells inthe mIL15⁺CAR⁺ T cell treatment group, but interestingly the T cellswere only found in the bone marrow (FIG. 41E) and may indicatepreferential homing or survival after tumor encounter. In a similarexperiment, survival was tested and mIL15⁺CAR⁺ T cell-treated miceexhibited significantly improved survival compared to the no T celltreatment or CAR⁺ T cell-treated mice [P=0.045 (mIL15⁺CAR⁺ T cellsversus CAR⁺ T cells, Log-rank Mantel-Cox test, n=7-8; FIG. 41).

In Vivo Persistence of mIL15⁺CAR⁺ T Cells in the Absence of CARActivation.

To assess if mIL15⁺CAR⁺ T cells can persist long-term in vivo withoutthe need for CAR signaling, modified T cells (mIL15^(+/−)CAR⁺ffLuc⁺)were infused into mice (with no tumor or exogenous cytokines) andmonitored for up to 47 days. Testing the T cells in this manner willalso elucidate whether the persisting T cells demonstrate unrestrictedgrowth or if they maintain their population in a homeostatic-like mannerNo CAR⁺ T cells were observed to persist, whereas the presence ofmIL15⁺CAR⁺ T cells exhibited sustained persistence in the absence ofexogenous cytokines and antigen (FIG. 42B). This was further confirmedby flow cytometry of CD3 stained cells isolated from the bone marrow,spleen, and peripheral blood (FIG. 43C). Assessing longitudinal T cellpersistence, flux values indicate that mIL15⁺CAR⁺ T cell levels appearedto level off over time or slowly decline, but did not indicate theoccurrence of uncontrolled expansion (FIG. 42D). These persisting Tcells were harvested and ex vivo expanded in the same manner aspreviously described for ex vivo expansion of genetically modified Tcells. These cells were capable of antigen-specific activation asassayed by interferon-γ production (FIG. 42E). This in vivo datademonstrates enhanced persistence of mIL15⁺CAR⁺ T cells in aCAR-independent and homeostatic-like manner whilst retaining theirantigen-specific responsiveness.

Cell Proliferation and Memory Kinetics in mIL15⁺CAR⁺ T Cells.

Flow cytometry analyses were used to further characterize mIL15⁺CAR⁺ Tcells (results shown in FIG. 35). Long-term persisting mIL15⁺CAR⁺ Tcells maintain in culture with low turnover rates as indicated byminimal PKH dilution over 10 days in samples that have been in constantculture for greater than one year. Moreover, dividing cells appeared tobe predominantly CCR7⁻. Such data indicates that the T cells aremaintained homeostatically as opposed to unrestricted autonomousproliferation or growth (FIG. 35A). The mIL15⁺CAR⁺ T cells in continuouslong-term culture (1.5 to 2.45 years) were activated with aAPCs,phenotyped and submitted for karyotyping. Phenotyping showed that theseT cells expressed mIL15 and karyoptyping results for all submitteddonors showed normal metaphase-spreads (FIG. 35B-C). FIG. 35D shows thememory kinetics after stimulation of cells using K562 aAPCs: Stim 1-CARversus mIL15⁺ CAR conditions during withdrawal: For these studies CAR Tcells got only IL-21 during the first 9 days of the stimulation and thenonly IL2 during Ag withdrawal conditions; mIL5⁺ CAR T cells got IL-21during the first 9 days and then no exogenous cytokines duringwithdrawal conditions. Results indicate that there is no difference inCCR7 and IL-7Ra expression at Day 19 post-Stim1 between CAR and mIL15⁺CAR T cells. CCR7 and IL-7Ra expression increases with time away fromantigen exposure (Day 29 vs Day 19) for mIL15-modified T cells only. CARexpression increases to ˜80% without exposure to antigen (PB522 was 51%and PB273 was 53% at 9 days post-Stim1. FIG. 35E, shows memory kinetics1 versus 2 stimulations of CAR and mIL15⁺ CAR T cells. Conditions duringwithdrawal: CAR T cells got only IL2 during Ag withdrawal which wasinstituted 10 days after the stimulation; mIL15⁺CAR T cells got noexogenous cytokines during withdrawal. Comparing CCR7 and IL-7Raexpression at similar time points after 1 Stim and 2 Stims indicated nomemory difference between CAR vs mIL15⁺ CAR with the 1 Stim at 19 days.In Stim 2, at equivalent time point to Stim 1 CAR T cells having 2 Stimshave far less CCR7 and almost no IL-7Ra compared to Stim 1 counterpartsmIL15⁺ CAR T cells having 2 Stims also have less memory, but retain more“memory” than CAR only T cells with mIL15⁺ CAR T cells having some CCR7left (but IL-7Ra is almost completely gone).

Example 8 Generation of Minimally Manipulated T-Cells Using mRNA CodingSB Transposase

Studies were undertaken to determine if use of a SB transposase providedas an mRNA for electroporation could further enhance CAR T cellproduction using a SB transposase system. For these studies cells wereelectroporated as detailed above using both a plasmid encoding a CARflanked by transposon repeats and a mRNA encoding the transposase (e.g.,SB11 or 100×). mRNA for the studies were m7GTP capped and included apoly-A tail. A schematic showing a protocol for CAR T cell productionusing a mRNA SB transposase provided as a mRNA is shown in FIG. 43. Forthese studies cells from donor #O were electroporated with the SB11mRNA, while studies cells from donor #1 were electroporated with the100× mRNA.

Cell produced from donor #O were characterized by flow cytometryanalysis for CAR expression and cell proliferation. As shown in FIG. 44Athe percentage of T-cells stably expressing CAR increases significantlyfrom day 9 (8.3%) to day 16 (66.2%). The T-cells grow quickly andamplify by 20-folds to day 16 after electroporation (FIG. 44B). Furtherstudies shown in FIG. 44D were used to assess the number of centralmemory T-cells (Tcm) at day 9 (left panel) and day 17 (right panel) postelectroporation. These results show that though the number of lessdifferentiated central memory T-cells (Tcm) declines between day 9 andday 17, it still remains relatively high (27%). Next, chromium releaseassays were used to determine the cytotoxic activity of the cells. Asshown in FIG. 44C the CAR T-cell produced provided CD-19-specificcytotoxicity against target cells, while essentially no cytotoxicactivity was seen for unmodified T-cells.

Cell produced from donor #1 were also studied. As shown in FIG. 45A theCAR T-cell produced from this donor also provided CD-19-specificcytotoxicity against target cells and essentially no cytotoxic activitywas seen for unmodified T-cells (right panels). Moreover, the modifiedT-cells kill CD19-positive target cells on day 9 and day 15 with similarefficiency despite the different number of CAR-positive cells. In FIG.45B the results of studies to assess CAR copy number and expression asshown. These results indicate that CAR DNA copy number decreases from1.5 to 0.9 from day 15 to day 22 and stays stable after that time point.

Finally, flow cytometry was used to assess cell viability followingelectroporation. As shown in FIG. 46, after electroporation withDNA/mRNA the total number of cells decreases first (day 1 and 2) andthen cells start to grow. According to the cellometer counts the cellnumber decrease by 59%-76% on day 2 after electroporation (viability24-41%).

Thus, the foregoing data demonstrated that by use of a CAR productiontechniques that employs mRNA encoding a transposase an effective numberof target-specific cytotoxic T cells can be produced in an extremelyshow period of time and with minimal manipulation. Although theefficiency of mRNA electroporation is donor-dependent, it appears thatthe SB100× transposase may be more efficient in CAR production.Interestingly, even though earlier after electroporation (day 9) thelower percentage of cells express CAR, the total population of cellskills targeted tumor cells almost as efficiently as one from the laterstage (day 15-16) and contains more central memory (less differentiated)cells. It was also confirmed that SB11 and SB100× coding mRNA provideintegration of about one copy CAR-coding gene per cell genome undertested conditions.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-77. (canceled)
 78. An isolated transgenic cell comprising anexpressed chimeric T-cell receptor (CAR) and an expressed membrane-boundIL-15, wherein the membrane-bound IL-15 comprises a fusion proteinbetween IL-15 and IL-15Ra.
 79. (canceled)
 80. The isolated cell of claim78, wherein the membrane-bound IL-15 comprises an amino acid sequence atleast 90% identical to SEQ ID NO:6.
 81. The isolated cell of claim 80,wherein the membrane-bound IL-15 comprises the amino acid sequence ofSEQ ID NO:6.
 82. The isolated cell of claim 78, comprising apolynucleotide sequence at least 90% identical to SEQ ID NO:
 7. 83. Theisolated cell of claim 82, comprising the polynucleotide sequence of SEQID NO:
 7. 84. The isolated cell of claim 78, wherein DNA encoding theCAR is integrated into the genome of the cell.
 85. The isolated cell ofclaim 78, wherein DNA encoding the membrane-bound IL-15 comprises anextra chromosomal element.
 86. The isolated cell of claim 78, whereinDNA encoding the membrane-bound IL-15 is integrated into the genome ofthe cell.
 87. A method of providing a T-cell response in a human subjecthaving a disease comprising administering an effective amount oftransgenic cells in accordance with claim 78 to the subject.
 88. Themethod of claim 87, wherein the disease is a cancer and wherein the CARis targeted to a cancer-cell antigen.
 89. The method of claim 88,wherein the subject has undergone a previous anti-cancer therapy. 90.The method of claim 89, wherein the subject is in remission.
 91. Themethod of claim 89, wherein the subject is free of symptoms of thecancer but comprises detectable cancer cells.
 92. A recombinantpolypeptide comprising an amino acid sequence at least 90% identical toSEQ ID NO:
 6. 93.-97. (canceled)
 98. The polypeptide of claim 92,comprising the amino acid sequence of SEQ ID NO:
 6. 99. A polynucleotideencoding a polypeptide at least 90% identical to SEQ ID NO:
 6. 100. Thepolynucleotide of claim 99 comprising a sequence at least 90% identicalto SEQ ID NO:
 7. 101. A host cell comprising a polynucleotide inaccordance with claim
 99. 102. The host cell of claim 101 wherein thecell is a T-cell, a T-cell precursor or an aAPC. 103.-104. (canceled)105. The method of claim 88, wherein the cancer-cell antigen is CD19.